Methods of identifying compounds that modulate IL-4 receptor-mediated IgE synthesis utilizing a chloride intracellular channel 1 protein

ABSTRACT

The present provides compounds capable of modulating IL-4 receptor-mediated IgE production, as well as IL-4 induced processes associated therewith, methods and kits for identifying such compounds that utilize a chloride intracellular channel 1 (CLIC1) as a surrogate analyte and methods of using the compounds in a variety of in vitro, in vitro and ex vivo contexts.

FIELD OF THE INVENTION

[0001] The present invention relates to compounds that modulateprocesses associated with isotype switching of B cells and IgEproduction, methods and kits for identifying such compounds and methodsof using such compounds in a variety of contexts, such as for thetreatment or prevention of diseases associated with or characterized byproduction and/or accumulation of IgE, including anaphylactichypersensitivity or allergic reactions, allergic rhinitis, allergicconjunctivitis, systemic mastocytosis, hyper IgE syndrome, and IgEgammopathies, atopic disorders such as atopic dermatitis, atopic eczemaand atopic asthma, and B-cell lymphoma

BACKGROUND OF THE INVENTION

[0002] The immune system protects the body against invasion by foreignenvironmental agents such as microorganisms or their products, foods,chemicals, drugs, molds, pollen, animal hair or dander, etc. The abilityof the immune system to protect the body against such foreign invadersmay be innate or acquired.

[0003] The acquired immune response, which stems from exposure to theforeign invader, is extremely complex and involves numerous types ofcells that interact with one another in myriad ways to express the fullrange of immune response. Two of these cell types come from a commonlymphoid precursor cell but differentiate along different developmentallines. One line matures in the thymus (T-cells); the other line maturesin the bone marrow (B-cells). Although T- and B-cells differ in manyfunctional respects, they share one of the important properties of theimmune response: they both exhibit specificity towards a foreign invader(antigen). Thus, the major recognition and reaction functions of theimmune response are contained within the lymph cells.

[0004] A third cell type that participates in the acquired immuneresponse is the class of cells referred to as antigen-presenting cells(APC). Unlike the T- and B-cells, the APC do not haveantigen-specificity. However, they play an important role in processingand presenting the antigen to the T-cells.

[0005] While the T- and B-cells are both involved in acquired immunity,they have different functions. Both T- and B-cells have antigen-specificreceptors on their surfaces that, when bound by the antigen, activatethe cells to release various products. In the case of B-cells, thesurface receptors are immunoglobulins and the products released by theactivated B-cells are immunoglobulins that have the same specificity forthe antigen as the surface receptor immunoglobulins. In the case ofactivated T-cells, the products released are not the same as theirsurface receptor immunoglobulins, but are instead other molecules,called cytokines, that affect other cells and participate in theelimination of the antigen. One such cytokine, released by a class ofT-cells called helper T-cells, is interleukin-4 (IL-4).

[0006] The immunoglobulins produced and released by B-cells must bind toa vast array of foreign invaders (antigens). All immunoglobulins sharecertain common structural features that enable them to: (1) recognizeand bind specifically to a unique structural feature on an antigen(termed an epitope); and (2) perform a common biological function afterbinding the antigen. Basically, each immunoglobulin consists of twoidentical light (L) chains and two identical heavy (H) chains. The Hchains are linked together via disulfide bridges. The portion of theimmunoglobulin that binds the antigen includes the amino-terminalregions of both L and H chains. There are five major classes of Hchains, termed α, δ, ε, γ and μ, providing five different isotypes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM. Although all five classesof immunoglobulins may possess precisely the same specificity for anantigen, they all have different biological functions.

[0007] While the immune system provides tremendous benefits inprotecting the body against foreign invaders, particularly those thatcause infectious diseases, its effects are sometimes damaging. Forexample, in the process of eliminating an invading foreign substancesome tissue damage may occur, typically as a result of the accumulationof immunoglobulins with non-specific effects. Such damage is generallytemporary, ceasing once the foreign invader has been eliminated.However, there are instances, such as in the case of hypersensitivity orallergic reactions, where the immune response directed against eveninnocuous agents such as inhaled pollen, inhaled mold spores, insectbite products, medications and even foods, is so powerful that itresults in severe pathological consequences or symptoms.

[0008] Such hypersensitivity or allergic reactions are divided into fourclasses, designated types I-IV. The symptoms of the type I allergicreactions, called anaphylactic reactions or anaphylaxis, include thecommon symptoms associated with mild allergies, such as runny nose,watery eyes, etc., as well as the more dangerous, and often fatal,symptoms of difficulty in breathing (asthma), asphyxiation (typicallydue to constriction of smooth muscle around the bronchi in the lungs)and a sharp drop in blood pressure. Also included within the class oftype I allergic reactions are atopic reactions, including atopicdermatitis, atopic eczema and atopic asthma.

[0009] Even when not lethal, such anaphylactic allergic reactionsproduce symptoms that interfere with the enjoyment of normal life. Oneneed only witness the inability of an allergy sufferer to mow the lawnor hike through the woods to understand the disruptive force even mildallergies have on everyday life. Thus, while the immune system is quitebeneficial, it would be desirable to be able to interrupt its responseto invading foreign agents that pose no risk or threat to the body.

[0010] IgE immunoglobulins are crucial immune mediators of suchanaphylactic hypersensitivity and allergic reactions, and have beenshown to be responsible for the induction and maintenance ofanaphylactic allergic symptoms. For example, anti-IgE antibodies havebeen shown to interfere with IgE function and alleviate allergicsymptoms (Jardieu, 1995, Curr. Op. Immunol. 7:779-782; Shields et al.,1995, Int. Arch. Allergy Immunol. 107:308-312). Thus, release and/oraccumulation of IgE immunoglobulins are believed to play a crucial rolein the anaphylactic allergic response to innocuous foreign invaders.Other diseases associated with or mediated by IgE production and/oraccumulation include, but are not limited to, allergic rhinitis,allergic conjunctivitis, systemic mastocytosis, hyper IgE syndrome, IgEgammopathies and B-cell lymphoma.

[0011] Although IgEs are produced and released by B-cells, the cellsmust be activated to do so (B-cells initially produce only IgD and IgM).The isotype switching of B-cells to produce IgE is a complex processthat involves the replacement of certain immunoglobulin constant (C)regions with other C regions that have biologically distinct effectorfunctions, without altering the specificity of the immunoglobulin. ForIgE switching, a deletional rearrangement of the IgH chain gene locusoccurs, which results in the joining of the switch region of the μ gene,Sμ, with the corresponding region of the ε gene, Sε.

[0012] This IgE switching is induced in part by IL-4 (or IL-13) producedby T-cells. The IL-4 induction initiates transcription through the Sεregion, resulting in the synthesis of germline (or “sterile”) εtranscripts (that is, transcripts of the unrearranged Cε H genes) thatlead to the production of IgE instead of IgM.

[0013] IL-4 induced germline ε transcription and consequent synthesis ofIgE is inhibited by interferon gamma (IFN-γ), interferon alpha (IFN-α)and tumor growth factor beta (TGF-β). In addition to the IL-4 signal, asecond signal, also normally delivered by T-cells, is required forswitch recombination leading to the production of IgE. This secondT-cell signal may be replaced by monoclonal antibodies to CD40,infection by Epstein-Barr virus or hydrocortisone.

[0014] Generally, traditional treatments for diseases mediated by IgEproduction and/or accumulation regulate the immune system followingsynthesis of IgE. For example, traditional therapies for the treatmentof allergies include anti-histamines designed to modulate theIgE-mediated response resulting in mast cell degranulation. Drugs arealso known that generally downregulate IgE production or that inhibitswitching of, but not induction of, germline ε transcription (see, e.g.,Loh et al., 1996, J. Allerg. Clin. Immunol. 97(5):1141).

[0015] Although these treatments are often effective, treatments thatact to reduce or eliminate IgE production altogether would be desirable.By reducing or eliminating IgE production, the hypersensitivity orallergic response may be reduced or eliminated altogether. Accordingly,the availability of compounds that are upstream modulators of IgEproduction, such as compounds capable of modulating, and in particularinhibiting, IL-4 receptor-mediated germline ε transcription, would behighly desirable.

[0016] The ability to screen for compounds capable of modulating IgEproduction, and in particular compounds that modulate IL-4 (or IL-13)induced germline ε transcription typically involves screening candidatecompounds in complex cell-based functional assays, such as thefunctional assays described in U.S. Pat. No. 5,958,707. These assaystypically involve contacting a cell comprising a reporter gene operablylinked to a promoter responsive to or inducible by IL-4 with a candidatecompound of interest in the presence of IL-4 and assessing the amount ofreporter gene product produced. The reporter gene is typically a genethat encodes a protein that produces an observable signal, such as afluorescent protein. The IL-4 inducible promoter may be a germline εpromoter. Compounds that antagonize (inhibit) IL-4 induced transcriptionwill yield reduced amounts of reporter gene product as compared tocontrol cells contacted with IL-4 alone. Compounds that agonize IL-4induced transcription will yield increased amounts of reporter geneproduct as compared to control cells contacted with IL-4 alone.Particularly useful functional assays for screening compounds for theability to modulate IL-4 inducible germline ε transcription aredescribed in U.S. Pat. No. 5,958,707, WO 99/58663 and WO 01/34806.

[0017] Although such functional screening assays are quite powerful andeffective, simpler assays that could be performed in cell-free systemsand/or that do not require a functional component, such as simplebinding assays with isolated proteins known to be involved in the IL-4signaling cascade responsible for the production of germline εtranscripts, and hence the production of IgE, would be beneficial.

SUMMARY OF THE INVENTION

[0018] These and other objects are furnished by the present invention,which in one aspect provides compounds, referred to herein as DL03compounds, which are capable of modulating, and in particularinhibiting, the IL-4 receptor-mediated signaling cascade involved inB-cell isotype switching to, and consequent production of, IgE. The DL03compounds of the invention are generally 8 to 30 amino acid residuepeptides or peptide analogs, or pharmaceutically-acceptable saltsthereof, characterized by structural formula (I):

Z¹-X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰-Z²

[0019] wherein:

[0020] X¹ is an aliphatic residue;

[0021] X² is an aromatic residue;

[0022] X³ is a small polar or small aliphatic residue;

[0023] X⁴ is a small polar or small aliphatic residue;

[0024] X⁵ is an aliphatic or nonpolar residue;

[0025] X⁶ is an aliphatic or nonpolar residue;

[0026] X⁷ is an aliphatic or nonpolar residue;

[0027] X⁸ is an aromatic residue;

[0028] X⁹ is small nonpolar residue;

[0029] X¹⁰ is an aliphatic or basic residue;

[0030] X¹¹ is a small polar or small aliphatic residue;

[0031] X¹² is a small polar or small aliphatic residue;

[0032] X¹³ is a small aliphatic or basic residue;

[0033] X¹⁴ is a small polar or small aliphatic residue;

[0034] X¹⁵ is a nonpolar or small aliphatic residue;

[0035] X¹⁶ is a small polar or small aliphatic residue;

[0036] X¹⁷ is a small aliphatic or basic residue;

[0037] X¹⁸ is a small aliphatic or conformationally-constrained residue;

[0038] X¹⁹ is a small aliphatic or aliphatic residue; and

[0039] X²⁰ is a small aliphatic or basic residue;

[0040] Z¹ is RRN—, RC(O)NR—, RS(O)₂NR— or an amino-terminal blockinggroup;

[0041] Z² is —C(O)OR, —C(O)O—, —C(O)NRR or a carboxyl-terminal blockinggroup;

[0042] each R is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl and substituted heteroarylalkyl;

[0043] each “˜” independently represents an amide, a substituted amideor an isostere of an amide;

[0044] each “—” represents a bond, or a 1 to 10 residue peptide orpeptide analog; and

[0045] wherein one or more of X¹, X², X¹⁹, or X²⁰ may be absent.

[0046] The DL03 compounds of the invention preferably have between 10and 25 residues; more preferably, the DL03 compounds of the inventionhave between 12 and 20 residues. In particular, the more preferredembodiments have 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues.

[0047] Particular DL03 compounds of the invention include DL03wt (LYTS ILLHGATTASMTKPLA), (SEQ ID NO:1) DL03IL (LYTSAALHGATTASMTKPLA), (SEQ IDNO:2) DL03KP (LYTS I LLHGATTASMTAALA), (SEQ ID NO:3) DL03LA (LYTS ILLHGATTASMTKPAR), (SEQ ID NO:4) DL03TS (LYAAI LLHGATTASMTKPLA), (SEQ IDNO:5) DL03GA (LYTS I LLHARTTASMTKPLA), (SEQ ID NO:6) DL03TT (LYTS ILLHGAAAASMTKPLA), (SEQ ID NO:7) DL03AS (LYTS I LLHGATTRAMTKPLA), (SEQ IDNO:8) DL03MT (LYTS I LLHGATTASAAKPLA). (SEQ ID NO:9)

[0048] The DL03 compounds of the invention inhibit IL-4 (or IL-13)induced germline ε transcription in cellular assays. As a consequence ofthis activity, the DL03 compounds of the invention can be used tomodulate the IL-4 receptor-mediated signaling cascade involved inisotype switching to, and consequent production of, IgE. In a specificembodiment, the DL03 compounds may be used to inhibit IL-4 (or IL-13)induced IgE production as a therapeutic approach towards the treatmentor prevention of diseases associated with, characterized by or caused byIgE production and/or accumulation, such as anaphylactichypersensitivity or allergic reactions and/or symptoms associated withsuch reactions, allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, and IgE gammopathies, atopic disorderssuch as atopic dermatitis, atopic eczema and atopic asthma, and B-celllymphoma.

[0049] The DL03 compounds of the invention can also be used in assays toidentify other compounds capable of effecting the above activities, aswill be described in more detail, below.

[0050] In addition to their ability to inhibit IL-4 (or IL-13) induciblegermline ε transcription, and hence IL-4 receptor-mediated IgEproduction, the DL03 compound of the invention also bind chlorideintracellular channel 1 (CLIC1). In particular, three DL03 compounds ofthe invention, DL03wt (LYTSILLHGATTASMTKPLA; SEQ ID NO: 1), peptideDL03IL (LYTSAALHGATTASMTKPLA; SEQ ID NO: 2), peptide DL03KP(LYTSILLHGATTASMTAALA; SEQ ID NO: 3) and peptide DL03LA(LYTSILLHGATTASMTKPAR; SEQ ID NO: 4) were found to bind human CLIC1(hCLIC1) (NCBI Sequence Database NP_(—)001279.2 (SEQ: ID NO: 12),corresponding nucleotide sequence at NM_(—)001288) in a yeast two hybridinteraction assay. Quite significantly, the ability of these DL03compounds to bind the hCLIC1 in this assay correlates with theirobserved ability to inhibit IL-4 (or IL-13) induced germline εtranscription. These observations provide the first evidence directlylinking CLIC1 to the IL-4 receptor-mediated signaling cascaderesponsible for isotype switching to, and consequent production of, IgE,and in particular as being an effector of germline ε transcription.Hence, these observations provide the first evidence directly linkingCLIC1 to the upstream regulation of isotype switching and/or IgEproduction.

[0051] This significant discovery enables the ability to use a CLIC1 asa “surrogate” analyte in screening assays to identify compounds thatmodulate or regulate the IL-4 receptor-mediated signaling cascadeleading to the production of IgE. Since induction of the ε promoter inresponse to IL-4 (or IL-13) is the first recognizable step necessary forisotype switching and consequent production of IgE, inhibition of IL-4(or IL-13) induced transcription of the ε promoter should preventB-cells from switching to and/or producing IgE. This significantdiscovery therefore permits the ability to use a CLIC1 as a surrogateanalyte in simple binding assays to identify compounds having a varietyof in vitro, in vivo and ex vivo therapeutic uses.

[0052] Accordingly, in another aspect, the invention provides methods ofidentifying compounds that modulate, and in particular inhibit, the IL-4receptor-mediated signaling cascade leading to the production of IgE.The methods generally comprise determining whether a candidate compoundof interest binds a CLIC1, wherein the ability to bind the CLIC1identifies the compound as being a modulator of the IL-4receptor-mediated signaling cascade leading to the production of IgE. Inone embodiment of the method, it is determined whether the candidatecompound competes for binding to the CLIC1 with a DL03 compound of theinvention, such as peptide DL03wt (SEQ ID NO: 1), peptide DL03IL (SEQ IDNO: 2), peptide DL03KP (SEQ ID NO: 3), or peptide DL03LA (SEQ ID NO: 4).In a specific embodiment of the method, compounds that inhibit IL-4 (orIL-13) induced germline ε transcription are identified. In a furtherembodiment, the methods comprise determining whether a candidatecompound of interest is a CLIC1 inhibitor.

[0053] In yet another aspect, the invention provides methods ofidentifying compounds that inhibit isotype switching of B-cells toproduce IgE and/or IgE production. The methods generally comprisedetermining whether a candidate compound of interest binds a CLIC1,wherein the ability to bind the CLIC1 identifies the compound as beingan inhibitor of isotype switching and/or IgE production. In oneembodiment of the method, it is determined whether the candidatecompound competes for binding to the CLIC1 with a DL03 compound of theinvention, such as peptides DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO:2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4).

[0054] In a specific embodiment of the method, compounds that inhibitIgE production are identified. In another specific embodiment, compoundsthat inhibit IL-4 receptor-mediated IgE production are identified. Instill another specific embodiment, compounds that inhibit CLIC1-mediatedIgE production are identified.

[0055] In still another aspect, the invention provides methods ofidentifying compounds that modulate, and in particular inhibit ordownregulate, processes mediated by or associated with IL-4receptor-mediated B-cell isotype switching and/or IgE production and/oraccumulation. Such processes include, but are not limited to,anaphylactic hypersensitivity or allergic reactions and/or symptomsassociated with such reactions, allergic rhinitis, allergicconjunctivitis, systemic mastocytosis, hyper IgE syndrome, and IgEgammopathies, atopic disorders such as atopic dermatitis, atopic eczemaand/or atopic asthma, and B-cell lymphoma. The methods generally involvedetermining whether a candidate compound of interest binds a CLIC1,where the ability to bind the CLIC1 identifies the compound as being amodulator of a process mediated by or associated with IL-4receptor-mediated isotype switching and/or IgE production and/oraccumulation. In one embodiment of the method, it is determined whetherthe candidate compound competes for binding the CLIC1 with a DL03compound of the invention, such as peptide DL03wt (SEQ ID NO: 1),peptide DL03IL (SEQ ID NO: 2), peptide DL03KP (SEQ ID NO: 3), or peptideDL03LA (SEQ ID NO: 4).

[0056] In yet another aspect, the invention provides methods ofidentifying compounds useful for treating disorders associated with, ormediated or caused by, IgE production and/or accumulation. The methodsgenerally comprise determining whether a candidate compound of interestbinds a CLIC1, wherein the ability to bind the CLIC1 identifies thecompound as being useful for treating disorders associated with, ormediated or caused by, IgE production and/or accumulation. In oneembodiment of the method, it is determined whether the candidatecompound competes for binding the CLIC1 with a DL03 compound of theinvention, such as peptide DL03wt (SEQ ID NO: 1), peptide DL03IL (SEQ IDNO: 2), peptide DL03KP (SEQ ID NO: 3), or peptide DL03LA (SEQ ID NO: 4).Diseases associated with, or mediated or caused by, IgE productionand/or accumulation for which therapeutic compounds may be identifiedaccording to the methods include, but are not limited to, anaphylactichypersensitivity or allergic reactions and/or symptoms associated withsuch reactions, allergic rhinitis, allergic conjunctivitis, systemicmastocytosis, hyper IgE syndrome, and IgE gammopathies, atopic disorderssuch as atopic dermatitis, atopic eczema and atopic asthma, and B-celllymphoma.

[0057] In another aspect, the invention provides compounds identified bythe various screening methods of the invention.

[0058] In still another aspect, the invention provides pharmaceuticalcompositions. The compositions generally comprise a DL03 compound of theinvention, a compound that competes for binding a CLIC1 with a DL03compound of the invention or a compound identified by the screeningmethods of the invention and a pharmaceutically-acceptable carrier,excipient or diluent.

[0059] In yet another aspect, the invention provides methods ofmodulating, and in particular inhibiting or downregulating, the IL-4receptor-mediated signaling cascade involved in B-cell isotype switchingto produce, and/or consequent production of, IgE, or processes involvedin this signal transduction cascade, such as IL-4 (or IL-13) inducedgermline ε transcription. The method generally involves administering toa cell a compound that binds a CLIC1 in an amount effective to modulatethis IL-4 receptor-mediated signaling cascade. In one embodiment of themethod, the compound inhibits IL-4 (or IL-13) induced germline εtranscription. In another specific embodiment, the compound inhibitsCLIC1-mediated germline ε transcription. The method may be practiced invitro, in vivo or ex vivo. In one embodiment, the cell is administered aDL03 compound of the invention, such as peptide DL03wt (SEQ ID NO: 1),peptide DL03IL (SEQ ID NO: 2), peptide DL03KP (SEQ ID NO: 3), peptideDL03LA (SEQ ID NO: 4), peptide DL03TS (SEQ ID NO: 5), peptide DL03GA(SEQ ID NO: 6), peptide DL03TT (SEQ ID NO: 7), peptide DL03AS (SEQ IDNO: 8), peptide DL03MT (SEQ ID NO: 9) or a compound that competes forbinding to a CLIC1 with an active DL03 compound of the invention.

[0060] In yet another aspect, the invention provides methods ofmodulating, and in particular inhibiting or downregulating, isotypeswitching to IgE and/or IgE production. The method generally involvesadministering to a cell an amount of a compound that binds a CLIC1effective to modulate isotype switching to IgE and/or IgE production.The method may be practiced in vitro, in vivo or ex vivo. In oneembodiment, the cell is administered a DL03 compound of the invention,such as peptide DL03wt (SEQ ID NO: 1), peptide DL03IL (SEQ ID NO: 2),peptide DL03KP (SEQ ID NO: 3), or peptide DL03LA (SEQ ID NO: 4), peptideDL03TS (SEQ ID NO: 5), peptide DL03GA (SEQ ID NO: 6), peptide DL03TT(SEQ ID NO: 7), peptide DL03AS (SEQ ID NO: 8), peptide DL03MT (SEQ IDNO: 9), or a compound that competes for binding to a CLIC1 with anactive DL03 compound of the invention.

[0061] In still another aspect, the invention provides methods oftreating or preventing diseases associated with, or mediated or causedby, IgE production and/or accumulation. The method generally comprisesadministering to an animal suffering from such a disease an amount of acompound that binds a CLIC1 effective to treat or prevent the diseaseand/or one or more of its symptoms. In one embodiment, the compoundadministered is a DL03 compound of the invention, such as peptide DL03wt(SEQ ID NO: 1), peptide DL03IL (SEQ ID NO: 2), peptide DL03KP (SEQ IDNO: 3), or peptide DL03LA (SEQ ID NO: 4), peptide DL03TS (SEQ ID NO: 5),peptide DL03GA (SEQ ID NO: 6), peptide DL03TT (SEQ ID NO: 7), peptideDL03AS (SEQ ID NO: 8), peptide DL03MT (SEQ ID NO: 9). In anotherembodiment, the compound administered is a compound that competes forbinding to a CLIC1 with an active DL03 compound of the invention.Diseases associated with, or mediated or caused by, IgE productionand/or accumulation that may be treated or prevented according to themethods of the invention include, but are not limited to, anaphylactichypersensitivity or allergic reactions (including food and drugallergies), and/or symptoms associated with such reactions, allergicrhinitis, allergic conjunctivitis, systemic mastocytosis, hyper IgEsyndrome, and IgE gammopathies, atopic disorders such as atopicdermatitis, atopic eczema and/or atopic asthma, and B-cell lymphoma. Themethod may be practiced therapeutically to treat the disease once theonset of the disease and/or its associated symptoms have alreadyoccurred, or prophylactically to prevent the onset of the disease and/orits associated symptoms. The methods may be practiced in veterinarycontexts or in the treatment of humans.

[0062] In a final aspect, the invention provides kits for carrying outthe various methods of the invention. In one embodiment, the kitcomprises a CLIC1 and a DL03 compound of the invention or a compoundthat competes for binding the CLIC1 with a DL03 compound of theinvention. The kit may further include additional components forcarrying out the methods of the invention, such as, by way of exampleand not limitation, buffers, labels and/or labeling reagents and/orinstructions teaching methods of using the kits.

BRIEF DESCRIPTION OF THE FIGURES

[0063]FIG. 1 illustrates the nucleotide sequence of a 603 bp fragment ofthe human germline ε promoter (SEQ ID NO: 11);

[0064]FIG. 2A provides a cartoon illustrating the diphtheria toxin (DT)selection of reporter cell line A5T4;

[0065]FIG. 2B provides a cartoon illustrating thetetracycline/doxycycline controlled peptide expression system ofreporter cell line A5T4;

[0066]FIG. 3 provides a cartoon illustrating the enrichment andscreening procedure used to identify certain DL03 compounds of theinvention;

[0067]FIG. 4 provides DNA transfer data for peptide DL03wt;

[0068]FIG. 5A provides a cartoon outlining a yeast two hybrid (YTH)screening assay used to identify potential binding partners or targetsfor peptide DL03wt;

[0069]FIG. 5B provides a cartoon illustrating a yeast two hybrid (YTH)assay used to identify hCLIC1 as a binding partner or target for peptideDL03wt;

[0070]FIG. 6 provides a cartoon summarizing strategies for reconfirmingpotential targets identified in the YTH assay depicted in FIG. 5A;

[0071]FIG. 7 provides interaction graphic profiles for peptide DL03wtand mutants derived therefrom;

[0072]FIG. 8 provides selection criteria for the interaction profilingmethod used to confirm hCLIC1 as a binding partner for peptide DL03wt;

[0073]FIG. 9 provides weighted graphic interaction/functional profilesfor peptide DL03wt and mutants derived therefrom.

[0074]FIG. 10 illustrates the amino acid sequence for human CLIC1 (SEQID NO: 10).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Abbreviations

[0076] The abbreviations used for the genetically encoded amino acidsare conventional and are as follows: Amino Acid Three-LetterAbbreviation One-Letter Abbreviation Alanine Ala A Arginine Arg RAsparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

[0077] When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D,” the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the N→C direction in accordancewith common convention.

[0078] The abbreviations used for the genetically encoding nucleosidesare conventional and are as follows: adenosine (A); guanosine (G);cytidine (C); thymidine (T); and uridine (U). Unless specificallydelineated, the abbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonulceosides on an individual basisor on an aggregate basis. When specified on an individual basis, theone-letter abbreviation is preceded by either a “d” or an “r,” where “d”indicates the nucleoside is a 2′-deoxyribonucleoside and “r” indicatesthe nucleoside is a ribonucleoside. For example, “dA” designates2′-deoxyriboadenosine and “rA” designates riboadenosine. When specifiedon an aggregate basis, the particular nucleic acid or polynucleotide isidentified as being either an RNA molecule or a DNA molecule.Nucleotides are abbreviated by adding a “p” to represent each phosphate,as well as whether the phosphates are attached to the 3′-position or the5′-position of the sugar. Thus, 5′-nucleotides are abbreviated as “pN”and 3′-nucleotides are abbreviated as “Np,” where “N” represents A, G,C, T or U. When nucleic acid sequences are presented as a string ofone-letter abbreviations, the sequences are presented in the 5→3′direction in accordance with common convention, and the phosphates arenot indicated.

[0079] Definitions

[0080] As used throughout the instant application, the following termsshall have the following meanings:

[0081] “Promoter” or “Promoter Sequence” refers to a DNA regulatoryregion capable of initiating transcription of a downstream (3′direction) coding sequence. A promoter typically includes atranscription initiation site (conveniently defined, for example, bymapping with nuclease S1) and protein binding domains responsible forbinding proteins that initiate transcription.

[0082] “TGF-β Inducible Promoter” refers to a promoter that initiatestranscription when a cell comprising a nucleic acid molecule includingsuch a promoter is exposed to, or contacted with, TGF-β. While notintending to be bound by any particular theory of operation, it isbelieved that contacting a cell comprising such a promoter with TGF-βcauses the activation of a DNA-binding protein that then binds the TGF-βinducible promoter and induces transcription of coding sequencesdownstream of the promoter

[0083] An “α promoter” or a “germline α promoter” is a TGF-β induciblepromoter that, when induced in a B-cell, leads to the production of IgAimmunoglobulins. Such germline α promoters are well-known in the art.Such promoters may be endogenous to a cell, or alternatively, they maybe engineered or exogenously supplied.

[0084] “IL-4 inducible promoter” refers to a promoter that initiatestranscription when a cell comprising a nucleic acid molecule includingsuch a promoter is exposed to, or contacted with, IL-4 or IL-13. Whilenot intending to be bound by any particular theory of operation, it isbelieved that contacting a cell comprising such a promoter with IL-4 (orIL-13) causes the activation of a DNA-binding protein that then bindsthe IL-4 inducible promoter and induces transcription of codingsequences downstream of the promoter.

[0085] An “ε promoter” or a “germline ε promoter” is an IL-4 induciblepromoter that, when induced in a B-cell, leads to the production of IgEimmunoglobulins. Such IL-4 inducible germline ε promoters are well-knownin the art. Such promoters may be endogenous to a cell or,alternatively, they may be engineered or supplied exogenously. Aspecific example of a germline ε promoter is the 603 bp IL-4 induciblefragment of the human ε promoter depicted in FIG. 1 (SEQ ID NO: 11).

[0086] A compound that “modulates an IL-4 inducible germline ε promoter”or that “modulates IL-4 induced germline ε transcription” has theability to change or alter expression downstream of the germline εpromoter induced by IL-4 (or IL-13). The change in IL-4 induceddownstream expression may occur at the mRNA (transcriptional) level orat the protein (translational) level. Hence, the change in IL-4 induceddownstream expression may be monitored at the RNA level, for example byquantifying induced downstream transcription products, or at the proteinlevel, for example by quantifying the amount or activity of induceddownstream translation products. The compound may act to modulate theIL-4 inducible germline ε promoter via any mechanism of action. Forexample, the compound may act to modulate the IL-4 inducible germline εpromoter by interacting with or binding a DNA binding protein involvedin the IL-4 induced transcription, or by interacting with or binding theIL-4 inducible germline ε promoter per se, or by interacting with orbinding to a molecule that functions in the signalling cascade triggeredby IL-4.

[0087] A compound that “modulates IL-4 receptor-mediated IgE productionand/or accumulation” has the ability to change or alter the amount ofIgE produced and/or accumulated by a B-cell activated via the IL-4receptor with IL-4 (or IL-13 or other IL-4 receptor ligand) and, in somecases, a second signal known to cause, in combination with IL-4 (orIL-13), isotype switching of B-cells to produce IgE. Such second signalmay be, for example, anti-CD40 monoclonal antibodies (anti-CD40 mAbs),infection by Epstein-Barr virus or hydrocortisone. The compound may actto modulate IL-4 receptor-mediated IgE production and/or accumulationvia any mechanism of action. For example, the compound may act tomodulate IL-4 induced germline ε transcription, and hence isotypeswitching, or the compound may act to modulate IgE production and/oraccumulation in an already switched cell. An “IL-4 induced” activityincludes activity (e.g., production of IgE, transcription of germline εpromoter isotype switching of B-cell) that is induced as a result of thebinding of IL-4, IL-13 or other IL-4 receptor ligand to the IL-4receptor.

[0088] “Identifying” in the context of screening assays meansdetermining whether a candidate compound unknown to possess a particularproperty of interest possesses the property of interest, as well asconfirming that a compound thought or known to possess a particularproperty of interest possesses the property of interest.

[0089] Compounds that “compete for binding with a DL03 compound” competefor binding to a CLIC1 (defined in a later section) with an active DL03compound of invention (described in more detail in a later section),such as peptide DL03wt (SEQ ID NO: 1), peptide DL03IL (SEQ ID NO: 2),peptide DL03KP (SEQ ID NO: 3), or peptide DL03LA (SEQ ID NO: 4), or withanother compound that competes for binding to a CLIC1 with an activeDL03 compound of the invention. For example, if compound 1 competes forbinding to a CLIC1 with an active DL03 compound and a candidate compoundcompetes for binding to the CLIC1 with compound 1, then for purposes ofthe present invention, the candidate compound competes for binding witha DL03 compound. Where competition with a specific category of compoundis intended, the modifiers “directly” and “indirectly” are used, where“directly” refers to competition with the stated compound and“indirectly” refers to competition with another compound that itselfcompetes with the stated compound.

[0090] “Alkyr” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic monovalenthydrocarbon group having the stated number of carbon atoms (i.e., C₁-C₆means from one to six carbon atoms) derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Theterm “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. The expression “lower alkyl” refersto alkyl groups composed of from 1 to 6 carbon atoms.

[0091] “Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl group. Typicalalkanyl groups include, but are not limited to, methanyl; ethanyl;propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

[0092] “Alkenyl” by itself or as part of another substituent refers toan unsaturated branched, straight-chain or cyclic alkyl group having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl , prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

[0093] “Alkynyl” by itself or as part of another substituent refers toan unsaturated branched, straight-chain or cyclic alkyl group having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl , etc.; and the like.

[0094] “Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene, etc. Typicalparent aromatic ring systems include, but are not limited to,aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like.

[0095] “Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonring atoms (i.e., C5-C₁₄ means from 5 to 14 carbon ring atoms) derivedby the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. Typical aryl groups include, but are notlimited to, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred. Particularly preferred aryls are cyclopentadienyl,phenyl and naphthyl.

[0096] “Arylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C₆-C₁₆) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₆) and the aryl moiety is (C₅-C₁₀). Inparticularly preferred embodiments the arylalkyl group is (C₆-C₁₃),e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is(C₁-C₁₃) and the aryl moiety is (C₅-C₁₀).

[0097] “Parent Heteroaromatic Ring System” refers to a parent aromaticring system in which one or more carbon atoms are each independentlyreplaced with the same or different heteroatoms or heteroatomic groups.Typical heteroatoms or heteroatomic groups to replace the carbon atomsinclude, but are not limited to, N, NH, P, O, S, Si, etc. Specificallyincluded within the definition of “parent heteroaromatic ring systems”are fused ring systems in which one or more of the rings are aromaticand one or more of the rings are saturated or unsaturated, such as, forexample, arsindole, benzodioxan, benzofuran, chromane, chromene, indole,indoline, xanthene, etc. Also included in the definition of “parentheteroaromatic ring system” are those recognized rings that includesubstituents, such as benzopyrone. Typical parent heteroaromatic ringsystems include, but are not limited to, arsindole, benzodioxan,benzofulran, benzopyrone, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

[0098] “Heteroaryl” by itself or as part of another substituent refersto a monovalent heteroaromatic group having the stated number of ringatoms (i.e., “5-14 membered” means from 5 to 14 ring atoms) derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred.

[0099] “Heteroarylalkyl” by itself or as part of another substituentrefers to an acyclic alkyl group in which one of the hydrogen atomsbonded to a carbon atom, typically a terminal or sp³ carbon atom, isreplaced with a heteroaryl group. Where specific alkyl moieties areintended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/orheterorylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is 1-6 membered and the heteroarylmoiety is a 5-14-membered heteroaryl. In particularly preferredembodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl,e.g., the alkanyl, alkenyl or alkynyl moiety is 1-3 membered and theheteroaryl moiety is a 5 -10 membered heteroaryl.

[0100] “Substituted Alkyl, Aryl, Arylalkyl, Heteroaryl orHeteroarylakyl” refers to an alkyl, aryl, arylalkyl, heteroaryl orheteroarylakyl group in which one or more hydrogen atoms is replacedwith another substituent group. Exemplary substituent groups include,but are not limited to, —OR′, —SR′, —NR′R—, —NO₂, —NO, —CN, —CF₃,halogen (e.g., —F, —Cl, —Br and —I), —C(O)R′, —C(O)OR′, —C(O)NR′,—S(O)₂R′, —S(O)₂NR′R′, where each R′ is independently selected from thegroup consisting of hydrogen and (C₁-C₆) alkyl.

[0101] The terms “percentage of sequence identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage may becalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package),or by visual inspection (see generally, Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, ajoint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1995 Supplement) (Ausubel)). Examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0102] While all of the above mentioned algorithms and programs aresuitable for a determination of sequence alignment and % sequenceidentity, for determination of % sequence identity in connection withthe present invention, the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.),using default parametersprovided, are preferred.

[0103] The DL03 Compounds

[0104] The DL03 compounds of the invention are generally peptides and/orpeptides analogs which, as will be discussed in more detail below, arecapable of modulating a variety of processes involved in IL-4receptor-mediated isotype switching of B-cells to produce IgE. The DL03compounds of the invention are generally peptides or peptide analogs, orpharmaceutically-acceptable salts thereof, that are from 8 to 30residues in length and include a “core” peptide or peptide analog,comprising at least 8 consecutive amino acid residues, preferably atleast 10 consecutive amino acid residues, more preferably at least 12amino acid residues, according to structural formula (II):

X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰

[0105] wherein:

[0106] X¹ is an aliphatic residue;

[0107] X² is an aromatic residue;

[0108] X³ is a small polar or small aliphatic residue;

[0109] X⁴ is a small polar or small aliphatic residue;

[0110] X⁵ is an aliphatic or nonpolar residue;

[0111] X⁶ is an aliphatic or nonpolar residue;

[0112] X⁷ is an aliphatic or nonpolar residue;

[0113] X⁸ is an aromatic residue;

[0114] X⁹ is a small nonpolar residue;

[0115] X¹⁰ is an aliphatic or basic residue;

[0116] X¹¹ is a small polar or small aliphatic residue;

[0117] X¹² is a small polar or small aliphatic residue;

[0118] X¹³ is a small aliphatic or basic residue;

[0119] X¹⁴ is a small polar or small aliphatic residue;

[0120] X¹⁵ is a nonpolar or small aliphatic residue;

[0121] X¹⁶ is a small polar or small aliphatic residue;

[0122] X¹⁷ is a small aliphatic or basic residue;

[0123] X¹⁸ is a small aliphatic or conformationally-constrained residue;

[0124] X¹⁹ is a small aliphatic or aliphatic residue; and

[0125] X²⁰ is a small aliphatic or basic residue.

[0126] The DL03 compounds of the invention include linear, branched andcyclic peptides and peptide analogs.

[0127] The DL03 compounds of the invention and/or the “core” peptides orpeptide analogs of structure (II) are defined, in part, in terms ofamino acids or residues bearing side chains belonging to certaindesignated classes. The definitions of the various classes of aminoacids or residues that define structure (II), and hence the DL03compounds of the invention, are as follows:

[0128] “Hydrophilic Amino Acid or Residue” refers to an amino acid orresidue having a side chain exhibiting a hydrophobicity of less thanzero according to the normalized consensus hydrophobicity scale ofEisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encodedhydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu(E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

[0129] “Acidic Amino Acid or Residue” refers to a hydrophilic amino acidor residue having a side chain exhibiting a pK value of less than about6 when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

[0130] “Basic Amino Acid or Residue” refers to a hydrophilic amino acidor residue having a side chain exhibiting a pK value of greater thanabout 6 when the amino acid is included in a peptide or polypeptide.Basic amino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-His (H), L-Arg (R) and L-Lys (K).

[0131] “Polar Amino Acid or Residue” refers to a hydrophilic amino acidor residue having a side chain that is uncharged at physiological pH,but which has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

[0132] “Hydrophobic Amino Acid or Residue” refers to an amino acid orresidue having a side chain exhibiting a hydrophobicity of greater thanzero according to the normalized consensus hydrophobicity scale ofEisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encodedhydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val(V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

[0133] “Aromatic Amino Acid or Residue” refers to a hydrophilic orhydrophobic amino acid or residue having a side chain that includes atleast one aromatic or heteroaromatic ring. The aromatic orheteroaromatic ring may contain one or more substituents such as —OH,—OR″, —SH, —SR″, —CN, halogen (e.g., —F, —Cl, —Br, —I), —NO₂, —NO, —NH₂,—NHR″, —NR″R″, —C(O)R″, —C(O)O⁻, —C(O)OH, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)NR″R″ and the like, where each R″ is independently (C₁-C₆) alkyl,substituted (C₁-C₆) alkyl, (C₂-C₆) alkenyl, substituted (C₂-C₆) alkenyl,(C₂-C₆) alkynyl, substituted (C₂-C₆) alkynyl, (C₅-C₁₀) aryl, substituted(C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, substituted (C₆-C₁₆) arylalkyl, 5-10membered heteroaryl, substituted 5-10 membered heteroaryl, 6-16 memberedheteroarylalkyl or substituted 6-16 membered heteroarylalkyl.Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y)and L-Trp (W). Although owing to the pKa of its heteroaromatic nitrogenatom L-His (H) is classified above as a basic residue, as its side chainincludes a heteroaromatic ring, it may also be classified as an aromaticresidue.

[0134] “Non-polar Amino Acid or Residue” refers to a hydrophobic aminoacid or residue having a side chain that is uncharged at physiologicalpH and which has bonds in which the pair of electrons shared in commonby two atoms is generally held equally by each of the two atoms (i.e.,the side chain is not polar). Genetically encoded non-polar amino acidsinclude L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

[0135] “Aliphatic Amino Acid or Residue” refers to a hydrophobic aminoacid or residue having an aliphatic hydrocarbon side chain. Geneticallyencoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L)and L-Ile (I).

[0136] The amino acid L-Cys (C) is unusual in that it can form disulfidebridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present invention L-Cys (C) is categorized as apolar hydrophilic amino acid, notwithstanding the generalclassifications defined above.

[0137] The amino acid Gly (G) is also unusual in that it bears no sidechain on its α-carbon and, as a consequence, contributes only a peptidebond to a particular peptide sequence. Moreover, owing to the lack of aside chain, it is the only genetically-encoded amino acid having anachiral α-carbon. Although Gly (G) exhibits a hydrophobicity of 0.48according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), for purposes of the present invention, Gly iscategorized as an aliphatic amino acid or residue.

[0138] Owing in part to its conformationally constrained nature, theamino acid L-Pro (P) is also unusual. Although it is categorized hereinas a hydrophobic amino acid or residue, it will typically occur inpositions near the N- and/or C-termini so as not to deleteriously affectthe structure of the DL03 compounds. However, as will be appreciated byskilled artisans, DL03 compounds may include L-Pro (P) or other similar“conformationally constrained” residues at internal positions.

[0139] “Small Amino Acid or Residue” refers to an amino acid or residuehaving a side chain that is composed of a total three or fewer carbonand/or heteroatoms (excluding the α-carbon and hydrogens). The smallamino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude Gly, L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S),L-Thr (T) and L-Asp (D).

[0140] “Hydroxyl-containing residue” refers to an amino acid containinga hydroxyl (—OH) moiety. Genetically-encoded hydroxyl-containing aminoacids include L-Ser (S) L-Thr (T) and L-Tyr (Y).

[0141] As will be appreciated by those of skill in the art, theabove-defined categories are not mutually exclusive. Indeed, thedelineated category of small amino acids includes amino acids from allof the other delineated categories except the aromatic category. Thus,amino acids having side chains exhibiting two or more physico-chemicalproperties can be included in multiple categories. As a specificexample, amino acid side chains having heteroaromatic moieties thatinclude ionizable heteroatoms, such as His, may exhibit both aromaticproperties and basic properties, and can therefore be included in boththe aromatic and basic categories. The appropriate classification of anyamino acid or residue will be apparent to those of skill in the art,especially in light of the detailed disclosure provided herein.

[0142] While the above-defined categories have been exemplified in termsof the genetically encoded amino acids, the DL03 compounds of theinvention are not restricted to the genetically encoded amino acids.Indeed, in addition to the genetically encoded amino acids, the DL03compounds of the invention may be comprised, either in whole or in part,of naturally-occurring and/or synthetic non-encoded amino acids. Certaincommonly encountered non-encoded amino acids of which the cycliccompounds of the invention may be comprised include, but are not limitedto: the D-enantiomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); omithine (Om); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(InAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thecompounds of the invention may be comprised will be apparent to those ofskill in the art (see, e.g., the various amino acids provided in Fasman,1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRCPress, Boca Raton, Fla., at pp. 3-70 and the references cited therein,all of which are incorporated by reference). These amino acids may be ineither the L- or D-configuration.

[0143] Those of skill in the art will recognize that amino acids orresidues bearing side chain protecting groups may also comprise the DL03compounds of the invention. Non-limiting examples of such protectedamino acids, which in this case belong to the aromatic category, include(protecting groups listed in parentheses), but are not limited to:Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl),Glu(δ-benzylester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom),His(benzyl), His(tos), Lys(finoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0144] Non-encoding amino acids that are conformationally constrained ofwhich the DL03 compounds of the invention may be composed include, butare not limited to, N-methyl amino acids (L-configuration);1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid;azetidine-3-carboxylic acid; homoproline (hPro); and1-aminocyclopentane-3-carboxylic acid.

[0145] The classifications of the genetically encoded and certain commonnon-encoded amino acids according to the categories defined above aresummarized in TABLE 1, below. It is to be understood that TABLE 1 is forillustrative purposes only and does not purport to be an exhaustive listof amino acids that can comprise the DL03 compounds of the invention.Other amino acids not specifically mentioned herein can be readilycategorized based on their observed physical and chemical properties inlight of the definitions provided herein. TABLE 1 Encoded and CertainCommon Non-Encoded Amino Acid Classifications Classification EncodedAmino Acids Non-encoded Amino Acids Hydrophobic Aromatic F, Y, W, H f,y, w, h, Phg, Nal, Thi, Tic, Pcf, Off, Mff, Pff, hPhe Non-Polar L, V, I,M, G, A, P l, v, i, m, g, a, p, Bua, Bug, MeIle, Nle, MeVal, Cha, MeGly,Aib Aliphatic A, V, L, I a, v, l, i, Dpr, Aib, Aha, MeGly, Bua, Bug,Mele, Cha, Nle, MeVal Hydrophilic Acidic D, E d, e Basic H, K, R h, k,r, Dpr, Gm, hArg, Paf, Dbu, Dab Polar C, Q, N, S. T c, q, n, s, t, Cit,AcLys, Mso, hSer Small G, A, V, C, N, S, T, D g, a, v, c, n, s, t, d

[0146] In the “core” peptides and peptide analogs of structure (II), thesymbol “˜” between each specified residue X″ designates a backboneconstitutive linking moiety. When the DL03 compounds of the inventionare peptides, each “˜” between the various X″ represents an amide orpeptide linkage of the following polarity: —C(O)—NH—. It is to beunderstood, however, that the DL03 compounds of the invention includeanalogs of peptides in which one or more amide or peptide linkages arereplaced with a linkage other than an amide or peptide linkage, such asa substituted amide linkage, an isostere of an amide linkage, or apeptido or amide mimetic linkage. Thus, when used in connection withdefining the various X″ comprising the DL03 compounds of the invention,the term “residue” refers to the C_(α) carbon and side chain moiety(ies)of the designated amino acid or class of amino acid. As a specificexample, defining X¹ as being a “Gly residue” means that X¹ is C_(α)H₂.Defining X¹ as being an “Ala residue” means that X¹ is C_(α)HCH₃ inwhich the C_(α) carbon is in either the D- or L-configuration. DefiningX¹ as being an “A residue” means that X¹ is C_(α)HCH₃ in which the C_(α)carbon is in the L-configuration.

[0147] Substituted amide linkages that may be included in the DL03compounds of the invention include, but are not limited to, groups ofthe formula —C(O)NR², where R² is (C₁-C₆) alkyl, (C₅-C₁₀) aryl,substituted (C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, substituted (C₆-C₁₆)arylalkyl, 5-10 membered heteroaryl, substituted 5-10 memberedheteroaryl, 6-16 membered heteroarylalkyl or substituted 6-16 memberedheteroarylalkyl. In a specific embodiment, R² is (C₁-C₆) alkanyl,(C₂-C₆) alkenyl, (C₂-C₆) alkynyl or phenyl.

[0148] Isosteres of amides that may be included in the DL03 compounds ofthe invention generally include, but are not limited to, —NR³—SO—,—NR³—S(O)₂—, —CH₂—CH₂—, —CH═CH— (cis and trans), —CH₂—NH—, —CH₂—S—,—CH₂—O—, —C(O)-CH₂—, —CH(OH)—CH₂— and —CH₂—S(O)₂—, where R³ is hydrogenor R² and R² is as previously defined. These interlinkages may beincluded in the DL03 compounds of the invention in either the depictedpolarity or in the reverse polarity. Peptide analogs including suchnon-amide linkages, as well as methods of synthesizing such analogs, arewell-known. See, for example, Spatola, 1983, “Peptide BackboneModifications,” In: Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Weinstein, Ed., Marcel Dekker, New York, pp. 267-357(general review); Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudson etal., 1979, Int. J. Prot. Res. 14:177-185 (—CH₂—NH—, —CH₂—CH₂); Spatolaet al., 1986, Life Sci. 38:1243-1249; Spatola, 1983, “Peptide BackboneModifications: the Ψ [CH₂S] Moiety as an Amide Bond Replacement,” In:Peptides: Structure and Function V, J. Hruby and D. H. Rich, Eds.,Pierce Chemical Co., Rockford, Ill., pp. 341-344 (—CH₂—S—); Hann, 1982,J. Chem. Soc. Parkin Trans. I. 1:307-314 (—CH═CH—, cis and trans);Almquist et al., 1980, J. Med. Chem. 23:1392-1398 (—C(O)—CH₂—); EuropeanPatent Application EP 45665; Chemical Abstracts CA 97:39405(—CH(OH)—CH₂—); Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404(—CH(OH)—CH₂—); and Hruby, 1982, Life Sci. 31:189-199 (—CH₂—S—).

[0149] Alternatively, one or more amide linkages may be replaced withpeptidomimetic and/or amide mimetic moieties. Non-limiting examples ofsuch moities are described in Olson et al., 1993, J. Med. Chem.36:3039-3049; Ripka & Rich, 1998, Curr. Opin. Chem. Biol. 2:441-452;Borchardt et al., 1997, Adv. Drug. Deliv. Rev. 27:235-256 and thevarious references cited therein.

[0150] While structure (II) contains 20 specified residue positions, itis to be understood that the DL03 compounds of the invention may containfewer than 20 residues. Indeed, truncated forms of structure (II)containing as few as 8 residues that retain one or more of the utilitiesdescribed herein are considered to be within the scope of the presentinvention. Truncated forms of the compounds of structure (II) areobtained by deleting one or more residues from either or both termini.Preferred truncated forms of structure (II) will contain at least 10residues; more preferred truncated forms of structure (II) will containat least 12 to at least 16 residues or more.

[0151] The core peptides or peptide analogs of structure (II) may alsobe extended at one or both termini. Typically, such extensions willrange from about 1 to about 5 residues, but may be even longer, so longas the compound retains one or more of the utilities described herein.For example, one or both termini may be extended by 6, 7, 8, 9, 10 oreven more residues.

[0152] In one embodiment of the invention, the extension has a sequencethat corresponds to a sequence of a signal peptide capable of effectingtransport across membranes, such that the DL03 compound is a “fusionpolypeptide.” Such fusion polypeptides are particularly advantageous foradministering to cells DL03 compounds of the invention that may notreadily traverse cell membranes. The signal sequence may be fused toeither the N-terminal or C-terminal portion of the DL03 compound,depending upon the characteristics of the particular signal sequenceselected. Signal sequences capable of transporting molecules into cellsare well-known in the art. Any of these sequences may be used inconnection with the DL03 compounds of the invention. Specific examplesof such sequences include HIV Tat sequences (see, e.g., Fawell et al.,1994, Proc. Natl. Acad. Sci. USA 91:664; Frankel et al., 1988, Cell55:1189; Savion et al., 1981, J. Biol. Chem. 256:1149; Derossi et al.,1994, J. Biol. Chem. 269:10444; Baldin et al., 1990, EMBO J. 9:1511;U.S. Patent No. 5,804,604; U.S. Pat. No. 5,670,617; and U.S. Pat. No.5,652,122, the disclosures of which are incorporated herein byreference), antennapedia sequences (see, e.g., Garcia-Echeverria et al.,2001, Bioorg. Med. Chem. Lett. 11:1363-1366; Prochiantz, 1999, Ann. NYAcad. Sci. 886:172-179; Prochiantz, 1996, Curr. Opin. Neurobiol.6:629-634; U.S. Pat. No. 6,080,724, and the references cited in all ofthe above, the disclosures of which are incorporated herein byreference) and poly(Arg) or poly(Lys) chains of 5-10 residues.Additional non-limiting examples of specific sequences can be found inU.S. Pat. No. 6,248,558; U.S. Pat. No. 6,043,339; U.S. Pat. No.5,807,746 U.S. Pat. No. 6,251,398; U.S. Pat. No. 6,184,038 and U.S. Pat.No. 6,017,735, the disclosures of which are incorporated herein byreference.

[0153] The terminus of the DL03 compounds of the invention correspondingto the amino terminus, if present, may be in the “free” form (e.g.,H₂N—), or alternatively may be acylated with a group of the formulaR2C(O)— or R²S(O)₂—, wherein R² is as previously defined. In oneembodiment, R² is selected from the group consisting of (C₁-C₆) alkyl,(C₅-C₁₀) aryl, (C₆-C₁₆) arylalkyl, 5-10 membered heteroaryl or 6-16membered heteroarylalkyl. In a specific embodiment, the R² group is agroup that facilitates entry of the DL03 compound into a cell. Suchgroups are well-known in the art.

[0154] In another embodiment, the amino terminus may be “blocked” with ablocking group designed to impart the DL03 compound with specifiedproperties, such as a low antigenicity. Non-limiting examples of suchblocking groups include polyalkylene oxide polymers such as polyethyleneglycol (PEG). A variety of polymers useful for imparting compounds, andin particular peptides and proteins, with specified properties are knownin the art, as are chemistries suitable for attaching such polymers tothe compounds. Specific non-limiting examples may be found in U.S. Pat.Nos. 5,643,575; 5,730,990; 5,902,588; 5,919,455; 6,113,906; 6,153,655;and 6,177,087, the disclosures of which are incorporated herein byreference.

[0155] Of course, skilled artisans will appreciate that any of thesetransport-effecting, acylating and/or blocking groups may also beattached to a side chain moiety of a DL03 compound. Residues havingappropriate functionalities for attaching such groups will be apparentto those of skill in the art, and include, by way of example and notlimitation, Cys, Lys, Asp and Glu.

[0156] The terminus of the DL03 compounds corresponding to theC-terminus, if present, may be in the form of an underivatized carboxylgroup, either as the free acid or as a salt, such as a sodium,potassium, calcium, magnesium salt or other salt of an inorganic ororganic ion, or may be in the form of a derivatized carboxyl, such as anester, thioester or amide. Such derivatized forms of the compounds maybe prepared by reacting a DL03 compound having a carboxyl terminus withan appropriate alcohol, thiol or amine. Suitable alcohols, thiols oramines include, by way of example and not limitation, alcohols of theformula R²OH, thiols of the formula R²SH and amines of the formulaR²NH₂, R²R²NH or NH₃, where each R² is, independently of the others, aspreviously defined.

[0157] The C-terminus may also include transport-effecting or otherblocking groups, such as those described above.

[0158] As will be recognized by skilled artisans, the various X″residues comprising the DL03 compounds of the invention may be in eitherthe L- or D-configuration about their C_(α) carbons. In one embodiment,all of the C_(α) carbons of a particular DL03 compound are in the sameconfiguration. In some embodiments of the invention, the DL03 compoundscomprise specific chiralities about one or more C_(α) carbon(s) and/orinclude non-peptide linkages at specified locations so as to impart theDL03 compound with specified properties. For example, it is well-knownthat peptides composed in whole or in part of D-amino acids are moreresistant to proteases than their corresponding L-peptide counterparts.Thus, in one embodiment, the DL03 compounds are peptides composed inwhole or in part of D-amino acids. Alternatively, DL03 compounds havinggood stability against proteases may include peptide analogs includingpeptide linkages of reversed polarity at specified positions. Forexample, DL03 compounds having stability against tryptic-like proteasesinclude peptide analogs having peptide linkages of reversed polaritybefore each L-Arg or L-Lys residue; DL03 compounds having stabilityagainst chymotrypsin-like proteases include peptide analogs havingpeptide linkages of reversed polarity before each small and medium-sizedL-aliphatic residue or L-non-polar residue. In another embodiment, DL03compounds having stability against proteases include peptide analogscomposed wholly of peptide bonds of reversed polarity. Other embodimentshaving stability against proteases will be apparent to those of skill inthe art. Additional specific embodiments of the DL03 compounds of theinvention are described below.

[0159] The DL03 compounds of the invention can be in a linear form or acyclic form, with or without branching. The cyclic forms can be cyclizedvia the terminal groups or via side chain groups on internal or terminalresidues, through covalent or non-covalent linkages. Additional linkinggroups may also be present to facilitate cyclization.

[0160] In one specific embodiment, the DL03 compounds of the inventionare 20-residue peptides or peptide analogs according to structuralformula (III):

Z³-X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰-Z⁴  (III)

[0161] wherein:

[0162] each X¹ through X²⁰ is as previously defined for structure (II);

[0163] Z³ is H₂N—, R⁴HN— or R⁴C(O)NH—;

[0164] Z⁴ is —C(O)O⁻, —C(O)OR⁴, —C(O)NHR⁴ or —C(O)NH₂;

[0165] each R⁴ is independently (C₁-C₆) alkyl or (C₁-C₆) alkanyl;

[0166] each “˜” is independently an amide linkage, a substituted amidelinkage or an isostere of an amide linkage; and

[0167] each “—” represents a bond.

[0168] In another specific embodiment, the DL03 compounds of theinvention are compounds according to structural formula (III) in whicheach “˜” is an amide linkage.

[0169] In yet another specific embodiment, the DL03 compounds of theinvention are compounds according to structural formula (III) in which:

[0170] X¹ is L-Leu;

[0171] X² is L-Tyr;

[0172] X³ is a small polar or small aliphatic residue;

[0173] X⁴ is a small polar or small aliphatic residue;

[0174] X⁵ is an aliphatic or nonpolar residue;

[0175] X⁶ is an aliphatic or nonpolar residue;

[0176] X⁷ is an aliphatic or nonpolar residue;

[0177] X⁸ is a L- His;

[0178] X⁹ is a small nonpolar residue;

[0179] X¹⁰ is an aliphatic or basic residue;

[0180] X¹¹ is a small polar or small aliphatic residue;

[0181] X¹² is a small polar or small aliphatic residue;

[0182] X¹³ is a small aliphatic or basic residue;

[0183] X¹⁴ is a small polar or small aliphatic residue;

[0184] X¹⁵ is a nonpolar or small aliphatic residue;

[0185] X¹⁶ is a small polar or small aliphatic residue

[0186] X¹⁷ is a small aliphatic or a basic residue;

[0187] X¹⁸ is a small aliphatic or a conformnationally-constrainedresidue;

[0188] X¹⁹ is a small aliphatic or an aliphatic residue; and

[0189] X²⁰ is a small aliphatic or a basic residue.

[0190] Preferably, X³ is L-Thr or L-Ala, X⁴ is L-Ser or L-Ala, X⁵ isL-Ile or L-Ala, X⁶ is L-Leu or L-Ala, X⁷ is L-Leu or L-Ala, X⁹ is L-Glyor L-Ala, X¹⁰ is L-Ala or L-Arg, X¹¹ is L-Thr or L-Ala, X¹² is L-Thr orL-Ala, X¹³ is L-Ala or L-Arg, X¹⁴ is L-Ser or L-Ala, X¹⁵ is L-Met orL-Ala, X¹⁶ is L-Thr or L-Ala, X¹⁷ is L-Lys or L-Ala, X¹⁸ is L-Pro orL-Ala, X¹⁹ is L-Leu or L-Ala and/or X²⁰ is L-Arg or L-Ala.

[0191] In another specific embodiment, the DL03 compounds of theinvention include compounds according to structural formula IV, andvariants of such compounds of formula IV in which 1, 2, 3, or 4,preferably 1 or 2, of the amino acid residues set forth in IV arereplaced by another amino acid selected from the same class (asdescribed herein) as the original amino acid or by an Ala or a Glyresidue:

Z¹-L˜Y˜T˜S˜I˜L˜L˜H˜G˜A˜T˜T˜A˜S˜M˜T˜K˜P˜˜L˜A-Z²   (IV)

[0192] wherein “—”, “˜”, Z¹ and Z² are as defined previously for formula(I).

[0193] In still another specific embodiment, the DL03 compounds of theinvention are selected from the group consisting of DL03wt(LYTSILLHGATTASMTKPLA(SEQ ID NO: 1)), DL03IL (LYTSAALHGATTASMTKPLA (SEQID NO: 2)), DL03KP (LYTSILLHGATTASMTAALA (SEQ ID NO: 3)), DL03LA(LYTSILLHGATTASMTKPAR (SEQ ID NO: 4)), DL03TS (LYAAI LLHGATTASMTKPLA(SEQID NO: 5)), DL03GA (LYTS ILLHARTTASMTKPLA (SEQ ID NO: 6)), DL03TT (LYTSI LLHGAAAASMTKPLA (SEQ ID NO: 7)) DL03AS (LYTS I LLHGATTRAMTKPLA (SEQ IDNO: 8)), DL03MT (LYTS I LLHGATTASAAKPLA (SEQ ID NO: 9)) and analogs andprotease-resistant analogs thereof.

[0194] Preferred DL03 compounds are selected from DL03wt (SEQ ID NO: 1),DL03IL (SEQ ID NO: 2), DL03KP (SEQ ID NO: 3)), DL03LA (SEQ ID NO: 4).

[0195] Active DL03 compounds of the invention are those that modulate,and in particular inhibit or downregulate, IL-4 induced IgE productionand/or accumulation and/or processes associated therewith. The DL03compounds of the invention may be assessed for such activity in anystandard assay that assesses the ability of a compound to modulate IL-4induced IgE production and/or accumulation. For example, a DL03 compoundof the invention may be administered to a human or animal B-cell (e.g.,primary B cells from blood, tonsils, spleens and other lymphoid tissues)stimulated with IL-4 (available from Pharmingen, Hamburg, Germany) andanti-CD40 mAbs (available from Ancell Corporation, Bayport Minn.) andthe amount of IgE produced measured, for example, by an ELISA technique,such as the ELISA technique described in Worm et al., 1998, Blood92:1713. The ELISA technique can use, for example, murine anti humanIgE, biotinylated anti human IgE and streptavidin biotinylatedhorseradish peroxidase complex. Specific ELISA assays and techniquesthat may be used are provided in the Examples section. Particular activeDL03 compounds include without limitation DL03wt (SEQ ID NO: 1), DL03IL(SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4), DL03TS(SEQ ID NO: 5), DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7) DL03AS (SEQID NO: 8), DL03MT (SEQ ID NO: 9).

[0196] For DL03 compounds that readily traverse cell membranes, thecompound may be administered to the cell by contacting the cell with thecompound. DL03 compounds composed wholly of genetically-encoded aminoacids that do not readily traverse cell membranes may be administered tothe cell using well-known delivery techniques. In one embodiment, suchDL03 compounds may be administered using well-known retroviral vectorsand infection techniques pioneered by Richard Mulligan and DavidBaltimore with Psi-2 lines and analogous retroviral packaging systemsbased upon NIH 3T3 cells (see Mann et al., 1993, Cell 33:153-159, thedisclosure of which is incorporated herein by reference). Suchhelper-defective packaging cell lines are capable of producing all ofthe necessary trans proteins (gag, pol and env) required for packaging,processing, reverse transcribing and integrating genomes. Those RNAmolecules that have in cis the ψ packaging signal are packaged intomaturing retrovirions. Virtually any of the art-known retroviral vectorsand/or transfection systems may be used. Specific non-limiting examplesof suitable transfection systems include those described in WO 97/27213;WO 97/27212; Choate et al., 1996, Human Gene Therapy 7:2247-2253;Kinsella et al., 1996, Human Gene Therapy 7:1405-1413; Hofmann et al.,1996, Proc. Natl. Acac. Sci. USA 93:5185-5190; Kitamura et al., 1995,Proc. Natl. Acac. Sci. USA 92:9146-9150; WO 94/19478; Pear et al., 1993,Proc. Natl. Acac. Sci. USA 90:8392-8396; Mann et aL, 1993, Cell33:153-159 and the references cited in all of the above, the disclosuresof which are incorporated herein by reference. Specific non-limitingexamples of suitable retroviral vector systems include vectors basedupon murine stem cell virus (MSCV) as described in Hawley et al., 1994,Gene Therapy 1:136-138; vectors based upon a modified MFG virus asdescribed in Rivere et al., 1995, Genetics 92:6733; pBABE as describedin WO 97/27213 and WO 97/27212; and the vectors depicted in FIG. 11 ofWO 01/34806, the disclosures of which are incorporated herein byreference. Other suitable vectors and/or transfection techniques arediscussed in connection with gene therapy administration, infra.

[0197] A specific assay for assessing IL-4 induced IgE production thatmay be used to assay DL03 compounds of the invention is described inWorm et al., 2001, Int. Arch. Allergy Immunol. 124:233-236. Generally, aDL03 compound modulates IL-4 induced IgE production if it yields anincrease or decrease in measured IgE levels of at least about 25% ascompared to control cells (i.e., cells activated with IL-4+ anti-CD40Mabs but not exposed to the DL03 compound). DL03 compounds that increaseIL-4 induced IgE production are IgE agonists whereas DL03 compounds thatdecrease IL-4 induced IgE production are IgE antagonists. Skilledartisans will appreciate that DL03 compounds that inhibit greater levelsof IL-4 induced IgE production, for example on the order of 50%, 60%,70%, 80%, 90%, or even more as compared to control cells, areparticularly desirable. Thus, while compounds that inhibit at leastabout 25% of IL-4 induced IgE production as compared to control cellsare active, compounds that inhibit at least about 50%, 75% or even moreIL-4 induced IgE production as compared to control cells are preferred.

[0198] In another embodiment, DL03 compounds may be assayed for theability to modulate IL-4 induced transcription of a germline ε promoter.Generally, such assays involve administering a DL03 compound to an IL-4induced cell comprising an IL-4 inducible gernline ε promoter andassessing the amount of gene expression (i.e. transcription) downstreamof the ε promoter. Depending upon the ability of the DL03 compound totraverse cell membranes, it may be administered to the cell bycontacting the cell with the compound or (for peptide compounds) via theretroviral transfection techniques described supra. The amount of thedownstream gene expression may be assessed at the mRNA level, forexample by quantifying the amount of a downstream transcription productproduced, or at the translation level, for example by quantifying theamount of a downstream translation product produced. In one embodiment,the germline ε promoter is operably linked to a reporter gene thatencodes a protein that produces an observable and/or detectable signal,such as a fluorescent protein. Specific examples of suitable assays forassessing DL03 compounds for the ability to modulate germline εtranscription are described in U.S. Pat. No. 5,958,707, WO 01/34806, WO99/58663, commonly owned copending application Ser. No. 09/712,821,filed Nov. 13, 2000 and commonly owned copending application Ser. No.09/076,624, filed May 12, 1998, the disclosures of which areincorporated herein by reference. Generally, a DL03 compound modulatesgermline ε transcription if it yields an increase or decrease inmeasured downstream expression of at least about 25% as compared tocontrol cells activated with IL-4 but not exposed to the DL03 compound.DL03 compounds that increase downstream expression are IL-4 agonists,whereas compounds that decrease (i.e. inhibit) downstream expression areIL-4 antagonists. Skilled artisans will appreciate that DL03 compoundsthat inhibit greater levels of IL-4 induced germline ε transcription,for example on the order of 50%, 60%, 70%, 80%, 90%, or even more ascompared to control cells, are particularly desirable. Thus, whilecompounds that inhibit at least about 25% of IL-4 induced germline εtranscription as compared to control cells are active, compounds thatinhibit at least about 50%, 75% or even more IL-4 induced germline εtranscription as compared to control cells are preferred. In oneembodiment of the invention, active DL03 compounds are those thatexhibit a reporter ratio of ≧1.1 in the A5T4 reporter line screeningassay described in the examples section. In general, the “reporterratio” is the ratio of the signal from a reporter under control of the εpromoter in the absence of a DL03 compound to that in the presence ofthe DL03 compound. In particular, for screening in the A5T4 reporterline that has been transformed with the inhibitor peptide vector, thereporter ratio can be determined from the ratio of the GFP fluorescenceof IL-4 stimulated cells in the presence of doxycycline or tetracycline(i.e., when expression of the peptide is repressed) to the GFPfluorescence of IL-4 stimulated cells in the absence of doxycycline ortetracycline.

[0199] As mentioned previously, B-cells initially produce IgD and IgMimmunoglobulins and, when induced by the proper cytokines, produce IgEs.B-cells can be induced to produce other types of immunoglobulins, suchas IgGs and IgAs, as well. For example, in the presence of the cytokineinterleukin-2 (IL-2), B-cells produce IgG1; in the presence of acombination of IL-2 and TGF-β, B-cells produce IgA. In many situations,it is desirable to selectively modulate (increase or decrease) theproduction of a single immunoglobulin isotype, as such specificitypermits the ability to treat or prevent diseases associated with theproduction and/or accumulation of the specified immunoglobulin isotypewithout affecting the immune system generally. Thus, in one embodiment,the DL03 compounds specifically modulate IL-4 induced germline εtranscription or IL-4 induced IgE production and/or accumulation. By“specific” is meant that the DL03 compound modulates IL-4 induced IgEproduction and/or accumulation or IL-4 induced germline ε transcriptionbut does not significantly affect the production and/or accumulation ofanother immunoglobulin, or transcription of the promoter of another Igisotype. In a particular embodiment, an DL03 compound specificallyinhibits IL-4 induced germline ε transcription or IL-4 induced IgEproduction or accumulation. Such DL03 compound does not significantlyinhibit production and/or accumulation of another Ig isotype, ortranscription of another Ig isotype promoter, if the observed inhibitionof the other Ig isotype in an appropriate assay is on the order of 10%or less as compared to control cells. Such specificity may be withrespect to a single Ig isotype, or may be with respect to one or more Igisotypes. For example, a DL03 compound may be assessed for specificityby assaying its ability to inhibit, for example, IgA production and/oraccumulation or to inhibit germline α transcription in assays similar tothose described above, except that the cells are activated witheffectors suitable for IgA switching and synthesis and amount of IgAproduced or the amount of expression downstream of a germline α promoteris assessed. Specific, non-limiting examples of DL03 compounds thatspecifically inhibit IL-4 induced IgE production and/or IL-4 inducedgermline ε transcription include peptides DL03wt (SEQ ID NO: 1), DL03IL(SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4) DL03wt (SEQID NO: 1), DL03IL (SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ IDNO: 4), DL03TS (SEQ ID NO: 5), DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO:7) DL03AS (SEQ ID NO: 8), and DL03MT (SEQ ID NO: 9).

[0200] As will be discussed in more detail below, it has been discoveredthat the ability of certain DL03 compounds to inhibit IL-4 induced IgEproduction and/or IL-4 induced germline ε transcription is mediated bybinding a CLIC1. Accordingly, DL03 compounds may also be assessed foractivity based upon their ability to bind a CLIC1 using, for example,any of the CLIC1 binding assays described infra. Generally, active DL03compounds are those having a binding constant (Kd) on the order of 10 mMor less, with Kds in the range of 100 μM, 10 ,μM, 1 μM, 100 nM, 10 nM, 1nM or even lower, being preferred. Alternatively, active DL03 compoundsare those that compete for binding a CLIC1 with another active DL03compound. In a specific embodiment, the DL03 compound competes forbinding a CLIC1 with peptides DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO:2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4), DL03TS (SEQ ID NO: 5),DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7) DL03AS (SEQ ID NO: 8), orDL03MT (SEQ ID NO: 9). The ability of a DL03 compound to compete forbinding a CLIC1 with another DL03 compound may be assessed usingconventional competitive binding assay techniques. Generally, activeDL03 compounds are those that exhibit an IC₅₀ in the range of 1 mM orlower, with IC₅₀s in the range of 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1nM or even lower, in such competitive binding assays being preferred.

[0201] Chemical Synthesis of the DL03 Compounds

[0202] DL03 compounds of the invention may be prepared using standardtechniques of organic synthesis. DL03 compounds that are peptides may beprepared using conventional step-wise solution or solid phase synthesis(see, e.g., Chemical Approaches to the Synthesis of Peptides andProteins, Williams et al., Eds., 1997, CRC Press, Boca Raton Fla., andreferences cited therein; FMOC Solid Phase Peptide Synthesis: APractical Approach, Chan & White, Eds., 2000, IRL Press, Oxford,England, and references cited therein).

[0203] Alternatively, DL03 compounds, may be prepared by way of segmentcondensation, as described, for example, in Liu et al., 1996,Tetrahedron Lett. 37(7):933-936; Baca et al., 1995, J. Am. Chem. Soc.117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and Tarn,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334. The condensation technique isparticularly useful for synthesizing DL03 compounds comprising Glyresidues. Other methods useful for synthesizing the DL03 compounds ofthe invention are described in Nakagawa et al., 1985, J. Am. Chem. Soc.107:7087-7092. DL03 compounds that are peptide analogs may besynthesized using the various methods described in the references citedin connection with amide isosteres and amide and peptidomimetics, supra.

[0204] DL03 compounds containing N- and/or C-terminal blocking groupscan be prepared using standard techniques of organic chemistry. Forexample, methods for acylating the N-terminus of a peptide or amidatingor esterifying the C-terminus of a peptide are well-known in the art.Modes of carrying other modifications at the N- and/or C-terminus willbe apparent to those of skill in the art, as will modes of protectingany side-chain functionalities as may be necessary to attach terminalblocking groups.

[0205] Formation of disulfide linkages, if desired, is generallyconducted in the presence of mild oxidizing agents. Chemical oxidizingagents may be used, or the compounds may simply be exposed toatmospheric oxygen to effect these linkages. Various methods are knownin the art, including those described, for example, by Tam et al., 1979,Synthesis 955-957; Stewart et al., 1984, Solid Phase Peptide Synthesis,2d Ed., Pierce Chemical Company Rockford, Ill.; Ahmed et al., 1975, J.Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides 1990164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. Anadditional alternative is described by Kamber et al., 1980, Helv. Chim.Acta 63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the DL03 compounds ofthe invention.

[0206] Cyclic peptides may be prepared or may result from the formationof single or multiple disulfide bonds, other side-chains or head-to-tailcyclizations, either directly or by way of an optional linker. Thecyclic peptides may be prepared using any art-known techniques for thepreparation of cyclic peptides and cyclic peptide analogs. For example,the peptide or peptide analog may be prepared in linear or non-cyclizedform using conventional solution or solid phase peptide and/or peptideanalog syntheses and cyclized using standard chemistries. The linearpolypeptides can be cyclized with a linking group between the twotermini, between one terminus and the side chain of an amino acid in thepeptide or peptide derivative, or between the side chains to two aminoacids in the peptide or peptide derivative. Suitable procedures forsynthesizing the peptide and peptide analogs described herein, as wellas suitable chemistries for cyclizing such compounds, are well known inthe art. For references related to synthesis of cyclic peptides thereader is referred to Tam et al., 2000, Biopolymers 52:311-332; Camameroet al, 1998, Angew. Chem. Intl. Ed. 37: 347-349; Tam et al., 1998, Prot.Sci. 7:1583-1592; Jackson et al., 1995, J. Am. Chem.Soc. 117:819-820;Dong et al., 1995, J. Am. Chem. Soc. 117:2726-2731; Ishida et al., 1995,J. Org. Chem. 60:5374-5375; WO 95/33765, published Jun. 6, 1995; Xue andDeGrado, 1994, J. Org. Chem. 60(4):946-952; Jacquier et al., 1991, In:Peptides 1990 221-222, Giralt and Andreu, Eds., ESCOM Leiden, TheNetherlands; Schmidt and Neubert, 1991, In: Peptides 1990 214-215,Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands; Toniolo, 1990,Int. J. Peptide Protein Res. 35:287-300; Ulysse et al., 1995, J. Am.Chem. Soc. 117:8466-8467; Durr et al., 1991, Peptides 1990 216-218,Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands; Lender et al.,1993, Int. J. Peptide Protein Res. 42:509-517; Boger and Yohannes, 1990,J. Org. Chem. 55:6000-6017; Brady et al., 1979, J. Org. Chem.4(18):3101-3105; Spatola et al., 1986, J. Am. Chem. Soc. 108:825-831;Seidel et aL, 1991, In: Peptides 1990 236-237, Giralt and Andreu, Eds.,ESCOM Leiden, The Netherlands; Tanizawa et al., 1986, Chem. Phar, Bull.34(10):4001-4011; Goldenburg & Creighton, 1983, J. Mol.Biol.165:407-413; WO 92/00995 and WO 94/15958. These methods may be routinelyadapted to synthesize the cyclic compounds of the invention and areincorporated into this application by reference.

[0207] Recombinant Synthesis of the DL03 Compounds

[0208] If the DL03 compound is composed entirely of genetically-encodedamino acids, or a portion of it is so composed, the peptide or therelevant portion may also be synthesized using conventional recombinantgenetic engineering techniques.

[0209] For recombinant production, a polynucleotide sequence encodingthe peptide is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The expression vehicle is then transfected into a suitable target cellwhich will express the peptide. Depending on the expression system used,the expressed peptide is then isolated by procedures well-established inthe art. Methods for recombinant protein and peptide production are wellknown in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. each of which is incorporated byreference herein in its entirety.)

[0210] To increase efficiency of production, the polynucleotide can bedesigned to encode multiple units of the peptide separated by enzymaticcleavage sites—either homopolymers (repeating peptide units) orheteropolymers (different peptides strung together) can be engineered inthis way. The resulting polypeptide can be cleaved (e.g., by treatmentwith the appropriate enzyme) in order to recover the peptide units. Thiscan increase the yield of peptides driven by a single promoter. In apreferred embodiment, a polycistronic polynucleotide can be designed sothat a single mRNA is transcribed which encodes multiple peptides (i.e.,homopolymers or heteropolymers) each coding region operatively linked toa cap-independent translation control sequence; e.g., an internalribosome entry site (IRES). When used in appropriate viral expressionsystems, the translation of each peptide encoded by the mRNA is directedinternally in the transcript; e.g., by the IRES. Thus, the polycistronicconstruct directs the transcription of a single, large polycistronicmRNA which, in turn, directs the translation of multiple, individualpeptides. This approach eliminates the production and enzymaticprocessing of polyproteins and may significantly increase yield ofpeptide driven by a single promoter.

[0211] Polynucleotides capable of generating or expressing certaincyclic peptide embodiments of the compounds of the invention may beprepared in vitro and/or in vivo. Polypeptides may be prepared frompolynucleotides to generate or express the cyclic peptides utilizing thetrans splicing ability of split inteins. Methods for making suchpolynucleotides to yield cyclic peptides are known in the art and aredescribed, for example, in WO 01/66565, WO 00/36093; U.S. patentapplication Ser. No. 60/358,827, entitled “Cyclic Peptides and AnalogsUseful to Treat Allergies”, filed on Feb. 21, 2002, the disclosures ofwhich are incorporated herein by reference.

[0212] A variety of host-expression vector systems may be utilized toexpress the peptides described herein. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

[0213] The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used inthe expression vector. For example, when cloning in bacterial systems,inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac(ptrp-lac hybrid promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

[0214] In cases where plant expression vectors are used, the expressionof sequences encoding the peptides of the invention may be driven by anyof a number of promoters. For example, viral promoters such as the 35SRNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al, 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, e.g., Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, N.Y., SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9.

[0215] In one insect expression system that may be used to produce thepeptides of the invention, Autographa californica, nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (e.g., see Smith et al., 1983, J. Virol. 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system may befound in Current Protocols in Molecular Biology, Vol. 2, Ausubel et al.,eds., Greene Publish. Assoc. & Wiley Interscience.

[0216] In mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

[0217] Other expression systems for producing DL03 peptides of theinvention will be apparent to those having skill in the art.

[0218] Purification of DL03 Compounds

[0219] The DL03 compounds of the invention can be purified by art-knowntechniques such as reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography and the like. The actual conditions used topurify a particular compound will depend, in part, on synthesis strategyand on factors such as net charge, hydrophobicity, hydrophilicity, etc.,and will be apparent to those having skill in the art.

[0220] For affinity chromatography purification, any antibody whichspecifically binds the compound may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a compound. Thecompound may be attached to a suitable carrier, such as BSA, by means ofa side chain finctional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0221] Monoclonal antibodies to a compound may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Kohler & Milstein, 1975,Nature 256:495-497 and/or Kaprowski, U.S. Pat. No. 4,376,110; the humanB-cell hybridoma technique described by Kosbor et al., 1983, ImmunologyToday 4:72 and/or Cote et aL., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030; and the EBV-hybridoma technique described by Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96. In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et aL.,1985, Nature 314:452-454; Boss, U.S. Pat. No. 4,816,397; Cabilly, U.S.Pat. No. 4,816,567) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Or“humanized” antibodies can be prepared (see, e.g., Queen, U.S. Pat. No.5,585,089). Alternatively, techniques described for the production ofsingle chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can beadapted to produce compound-specific single chain antibodies.

[0222] Antibody fragments which contain deletions of specific bindingsites may be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)2 fragments, which can be producedby pepsin digestion of the antibody molecule and Fab fragments, whichcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor the peptide of interest.

[0223] The antibody or antibody fragment specific for the desiredpeptide can be attached, for example, to agarose, and theantibody-agarose complex is used in immunochromatography to purifypeptides of the invention. See, Scopes, 1984, Protein Purification:Principles and Practice, Springer-Verlag New York, Inc., N.Y.,Livingstone, 1974, Methods In Enzymology: Immunoaffinity Chromatographyof Proteins 34:723-731.

[0224] As will be recognized by skilled artisans, the above methods mayalso be used to prepare anti-CLIC1 antibodies. Such anti-CLIC1antibodies may be used in the various methods described herein, forexample, to inhibit IL-4 induced isotype switching and/or IgE productionand/or to inhibit IL-4 induced germline ε transcription. Such antibodiesmay also be used in the various therapeutic methods described herein.

[0225] Screening Assays

[0226] The present inventors have discovered that the IgE regulatoryeffects of four DL03 compounds of the invention, peptides DL03wt (SEQ IDNO: 1), DL03IL (SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), and DL03LA (SEQ IDNO: 4), are mediated by a Chloride intracellular channel 1 (CLIC1) (NCBISequence Database NP_(—)001279.2 (SEQ ID NO: 12)). Specifically, thepresent inventors have discovered that these four DL03 compounds bind ahuman CLIC1 in yeast two hybrid (YTH) assays in a manner that correlateswith their ability to inhibit IL-4 induced germline ε transcription inin vitro cellular assays.

[0227] Chloride ion channels in plasma membranes play important roles inthe maintaining cell volume, transepithelial transport, setting themembrane potential, bone resorption, and in the response to certainneurotransmitters (Jentsch, 1994, Curr. Opin. Cell Biol. 6, 600-601;Jentsch et al., 1997, Bioessays 19, 117-126). Chloride ion channels arealso present in intracellular membranes and play important roles inacidification of intracellular compartments and in exocytosis(Al-Awqati,1995, Curr. Opin. Cell. Biol. 7, 504-508; 4. Redhead et al, 1997, Mol.Biol. Cell 8, 691-704).

[0228] Four structurally unrelated types of chloride ion channels havebeen identified: 1.) the ligand-gated family (e.g. γ-aminobutyric acidand glycine receptors) (Hosie et al., 1997, Trends Neurosci. 20,578-583); 2.) the cystic fibrosis transmembrane conductance regulator(CFTR), a member of the ATP binding cassette family of proteins (Seibertet al., 1997, J. Bioenerg. Biomembr. 29, 429-442) 3.) the chloride ionchannels (ClC) family (Jentsch et al., 1995, J. Physiol. (Lond.) 482,19-25) and 4.) the chloride intracellular channels (CLIC) family(Edwards, 1999, Am. J. Physiol. 276, F398-F408).

[0229] The CLIC family is defined by a COOH-terminal core segment of˜230 amino acids that is highly conserved among all family members. Todate there are at least seven members of the CLIC family: CLIC1 (NCC27)(Valenzuela et al., 1997, J. Biol. Chem. 272, 12575-12582), CLIC2 (Heisset al., 1997, Genomics 45, 224-228), CLIC3 (Qian et al., 1999, J. Biol.Chem. 274, 1621-1627), CLIC4 (Duncan et al, 1997, J. Biol. Chem. 272,23880-23886), CLIC5 (Berryman et al., 2000, Mol. Biol. Cell 11,1509-1521), p64 (Landry et al.,1993, J. Biol. Chem. 268, 14948-14955),and parchorin (Nishizawa et al., 2000, J. Biol. Chem. 275, 11164-11173).

[0230] The first member of CLIC family described was the bovineintracellular chloride channel p64 (Landry et al. 1993, supra). Ahomologue of p64 was isolated from rat brain tissue, and designatedp64H1 (Duncan et al. 1997, supra). CLIC1 isolated from the humanmonocyte cell line U937, is a much smaller protein than p64, containingonly 241 amino acids. CLIC1 shares 63% identity with bovine p64, 61%identity with human CLIC2, 66% with rat p64H1 and 49% identity withhuman CLIC3 (Valenzuela et al., 2000, J Phys. 529.3, 541-552), 67%identity with human CLIC4 (NCBI: NP_(—)039234; gi:7330335) and 63%identity with rat CLIC5 (NCBI: NP_(—)446055.11 gi:6758390).

[0231] The CLIC1 gene is highly conserved across species. A CLIC1 cDNAprobe cross hybridizes with genomic DNA from all the eukaryotic speciesstudied, including monkey, rat, mouse, dog, cow, rabbit and yeast. CLIC1shares 90-100% identity to a growing number of human, rat and mouseexpressed sequence tags (EST). CLIC1 is 99-100% identical over 109 aminoacids to a porcine EST (SSC24B10) and 63% identical over 152 amino acidsto an EST from zebrafish (AA497337).

[0232] The CLIC1 protein is a 27 kDa chloride ion channel that exists incells as a soluble cytoplasmic protein and an integral membrane protein.In phospholipid membranes, CLIC1 functions as an anion selective channel(Tulk et al. (2002) Am. J. Physiol. 282(5):C1103-12). Within the CLICfamily, only CLIC1 and CLIC3 are dominantly nuclear in distribution(Qian et al., 1999, supra). The structure of the soluble form of CLIC1has been determined at 1.4-Å resolution (Harrop et al., 2001, J. Biol.Chem., Vol. 276, 48, 44993-45000). The soluble form of CLIC1 ismonomeric, although a recent publication (Warton et al. 2002 J. BiolChem. epublished Apr. 26, 2002) suggests that CLIC1 in ion channelsexists in a tetrameric assembly of subunits. CLIC1 is structurallyhomologous to the glutathione S-transferase superfamily, and it has aredox-active site resembling glutaredoxin (Harrop et al., 2001, supra).The structure of the complex of CLIC1 with glutathione shows thatglutathione occupies the redox-active site, which is adjacent to anopen, elongated slot lined by basic residues. The structure indicatesthat CLIC1 is likely to be controlled by redox-dependent processes(Harrop et al., 2001, supra).

[0233] Although the precise function of CLIC1 remains unclear, it hasbeen implicated in the regulation of the cell cycle (Valenzuela et al.,2000, supra). Electrophysiological studies in Chinese hamster ovary(CHO-K1) cells indicated that CLIC1 chloride conductance variedaccording to the stage of the cell cycle, being expressed only on theplasma membrane of cells in G2/M phase. (Valenzuela et al., 2000,supra). Cl⁻ ion channel blockers known to block CLIC1 led to arrest ofCHO-K1 cells in the G2/M stage of the cell cycle, the same stage atwhich this ion channel is selectively expressed-on the plasma membrane(Valenzuela et al., 2000, supra). A protein homologous to CLIC1, DBP-1,was recently identified by its ability to bind to certaindiarylsulfonylureas (DASUs) (US2002/0034764A1, EP0987552A2). As DASUsare known to inhibit the production of inflammatory cytokines like IL-1and IL-18, DBP-1 has been proposed as a target for screening compoundsuseful for treatment of inflammatory disorders including asthma.However, the present inventors are the first to discover a link betweenhCLIC1 and modulation of the IL-4 signaling cascade involved in theproduction of IgE, and in particular to IL-4 induced isotype switchingof B-cells to produce IgE.

[0234] This significant discovery enables, for the first time, theability to use a CLIC1 as a “surrogate” analyte in simple binding assaysto screen for and/or identify compounds involved in IL-4 induced IgEregulation. Such compounds are useful in the treatment and/or preventionof diseases caused by or associated with IgE production and/oraccumulation, such as anaphylactic hypersensitivity or allergicreactions and or symptoms associated with such reactions, allergicrhinitis, allergic conjunctivitis, systemic mastocytosis, hyper IgEsyndrome and IgE gammopathies, and atopic disorders such as atopicdermatitis, atopic eczema and atopic asthma, and B-cell lymphoma.

[0235] Thus, the invention also provides methods and kits useful foridentifying compounds having specified utilities. In specificembodiments, the methods and kits may be used to identify compounds thatinhibit IL-4 induced IgE production and/or accumulation, compounds thatinhibit IL-4 induced isotype switching of B-cells to produce IgE,compounds that inhibit IL-4 induced germline ε transcription, and/orcompounds useful to treat or prevent diseases caused by or associatedwith IgE production and/or accumulation, such as those described above.

[0236] “Chloride intracellular channel 1” or “CLIC1” useful in thescreening methods and kits of the invention include any proteinrecognized in the art as belonging to the CLIC1 family. In particular,useful CLIC1s include human CLIC1(“hCLIC1”) (NP_(—)001279.2,XP_(—)004183.3, sp O00299) shown in FIG. 10 as SEQ ID NO: 10, andallelic and species variants thereof, as well as fragments and fusionsthereof that bind to the DL03 compounds, particularly DL03wt. Typically,such proteins will have polypeptide sequences that share at least about80% identity at the amino acid level with hCLIC1. Preferably, the CLIC1protein employed in the methods of the present invention will have atleast 85%, 90%, 95% or even higher % identity with hCLIC1. Specificexamples of CLIC1s suitable for use in the methods and kits of theinvention include CLIC1 derived from humans (Valenzuela et al., 1997,supra)as well as the various corresponding mammalian homologs thereof(for example, mouse, rabbit, ect.). The amino acid sequences of thesevarious CLIC1s, as well as the sequences of nucleic acid moleculesencoding these CLIC1s, are known in the art and can be found in thefollowing references and/or NCBI (GenBank) entries: Mus musculus CLCP(gi|3986758|gb|AAC84155.1; Oryctolagus cuniculus chloride intracellularchannel protein (gil|4572050|gb|AAK67356.1|AF387765_(—)1).

[0237] As will be recognized by skilled artisans, mutants and/orfragments of a CLIC1 may also be used in the assays and kits of theinvention. Mutants and/ or fragments of CLIC1 that are useful in thisregard are those mutants and/or fragments that retain the ability tobind an active DL03 compound, preferably peptide DL03wt (SEQ ID NO: 1),peptide DL03IL (SEQ ID NO: 2), peptide DL03KP (SEQ ID NO: 3), or peptideDL03LA (SEQ ID NO: 4). Suitable fragments include CLIC1s that aretruncated at the N- and/or C-terminus by one or more amino acids,typically by about 1 to 10-20 amino acids, although fragments truncatedby more amino acids may be used, provided the fragments bind an activeDL03 compound. Additionally, mutants and/or fragments of CLIC1 thatsubstantially retain one or more of the biological activities of CLIC1are useful in the assays and kits of the invention. By “substantiallyretain” is meant that the mutant or fragment has at least 10% of thebiological activity of CLIC1 as measured by any conventional assay ofCLIC1 activity; preferably, the mutant or fragment has at least 50% ofthe biological activity of CLIC1.

[0238] CLIC1 mutants useful in the methods and kits of the inventioninclude conservative mutants in which one or more amino acids isreplaced with another amino acid of the same class, as defined above inconnection with the description of the DL03 compounds. Of course CLIC1mutants including non-conservative substitutions may also be used, solong as the particular mutant binds an active DL03 compound and/orsubstantially retains CLIC1 activity. Thus, unless indicated otherwise,the expression “chloride intracellular channel 1” or “CLIC1” as usedherein specifically includes such mutants and/or fragments in additionto the full-length wild-type proteins.

[0239] The CLIC1s may be obtained using conventional recombinant andpurification techniques or may be isolated directly from the naturalsource. For example, any of the recombinant techniques discussed suprain connection with the DL03 compounds may be used to produce a CLIC1suitable for use in methods and kits of the invention. Suchrecombinantly-produced CLIC1 s may be isolated using affinitychromatography (for example with an anti-CLIC1 antibody or otherconventional techniques. Specific examples that may be routinely adaptedare described in Tulk et al., Am J Physiol Cell Physiol. (2002)282(5):C1103-12; Harrop et al., 2001, supra; Tonini et al., FASEB J.June 2000;14(9):1171-8; Tulk et al., J Biol Chem. (2000) September1;275(35):26986-93, the disclosures of which are incorporated herein byreference. Other techniques for obtaining CLIC1s for use in the methodsand kits of the invention will be apparent to those of skill in the art.

[0240] Any screening technique known in the art for determining whethercompounds bind one another can be used to screen for compounds that binda CLIC1. The compounds screened can range from small organic moleculesto large polymers and biopolymers, and can include, by way of exampleand not limitation, small organic compounds, saccharides, carbohydrates,polysaccharides, lectins, peptides and analogs thereof, polypeptides,proteins, antibodies, oligonucleotides, polynucleotides, nucleic acids,etc. In one embodiment, the candidate compounds screened are smallorganic molecules having a molecular weight in the range of about100-2500 daltons. Such candidate molecules will often comprise cyclicalstructures composed of carbon atoms or mixtures of carbon atoms and oneor more heteroatoms and/or aromatic, polyaromatic, heteroaromatic and/orpolyaromatic structures. The candidate agents may include a wide varietyof functional group substituents. In one embodiment, the substituent(s)are independently selected from the group of substituents known tointeract with proteins, such as, for example, amine, carbonyl, hydroxyland carboxyl groups.

[0241] The candidate compounds may be screened on a compound-by-compoundbasis or, alternatively, using one of the myriad library techniquescommonly employed in the art. For example, synthetic combinatorialcompound libraries, natural products libraries and/or peptide librariesmay be screened using the assays of the invention to identify compoundsthat bind a CLIC1. The candidate compounds may be assessed for theability to bind a CLIC1 per se, or they may be assessed for the abilityto competitively bind a CLIC1 in the presence of an active DL03 compoundof the invention, such as peptide DL03wt (SEQ ID NO: 1), peptide DL03IL(SEQ ID NO: 2), peptide DL03KP (SEQ ID NO: 3), or peptide DL03LA (SEQ IDNO: 4), or another compound that competitively binds a CLIC1 in thepresence of an active DL03 compound of the invention. These competitivebinding assays can identify compounds that bind the CLIC1 atapproximately the same site as the active DL03 compound. Myriadtechniques for carrying out competitive binding assays are known in theart. Any of these techniques may be employed in the present invention.

[0242] Such binding experiments may be conducted wholly in solution or,alternatively, either the CLIC1 or the candidate compound may beimmobilized on a solid support. For example, the CLIC1 or the candidatecompound may be attached to a glass or other bead or a solid surfacesuch as, for example, the bottom of a petri dish. The immobilization maybe mediated by non-covalent interactions or by covalent interactions.Methods for immobilizing myriad types of compounds and proteins on solidsupports are well-known. Any of these methods may be used to immobilizethe CLIC1 and/or candidate compound on solid supports.

[0243] The binding assays may employ a purified CLIC1 or, alternatively,the assays may be carried out with nucleosol and/or cytosol fractionsfrom cells that express the CLIC1, either endogenously or recombinantly.

[0244] Whether carried out in solution or with an immobilized CLIC1 orcandidate compound, the CLIC1 and candidate compound are typicallycontacted with one another under conditions conducive to binding.Although the actual conditions used can vary, typically the bindingassays are carried out under physiological conditions. Theconcentrations of CLIC1 and candidate compound used will depend upon,among other factors, whether the CLIC1 or candidate compound isimmobilized or free in solution, the binding affinities of candidatecompounds, etc. Actual concentrations suitable for a particular assaywill be apparent to those of skill in the art.

[0245] In many embodiments of the kits and assays of the invention itmay be convenient to employ a labeled CLIC1 and/or labeled candidatecompound. For example, in one convenient embodiment, binding is assessedby contacting an immobilized candidate compound with a labeled CLIC1 andassaying for the presence of immobilized label. For such embodiments,the label may be a direct label, i.e., a label that itself is detectableor produces a detectable signal, or it may be an indirect label, i.e., alabel that is detectable or produces a detectable signal in the presenceof another compound. The method of detection will depend upon thelabeled used, and will be apparent to those of skill in the art.

[0246] Examples of suitable direct labels include radiolabels,fluorophores, chromophores, chelating agents, particles,chemiluminescent agents and the like. Suitable radiolabels include, byway of example and not limitation, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I and ¹⁸⁶Re. A suitable method for carrying outbinding assays with a chloride intracellular channel or biologicallyactive fragments thereof is described in U.S. Pat. No. 6,228,616B1, thedisclosure of which is incorporated herein by reference. Suitablefluorophores include, by way of example and not limitation, fluorescein,rhodamine, phycoerythrin, Texas red, free or chelated lanthanide seriessalts such as Eu³⁺ and the myriad fluorophores available from MolecularProbes Inc., Eugene, Oreg. Examples of suitable colored labels include,by way of example and not limitation, metallic sol particles, forexample, gold sol particles such as those described by Leuvering (U.S.Pat. No. 4,313,734); dye sole particles such as described by Gribnau etal. (U.S. Pat. No. 4,373,932) and May et al. (WO 88/08534); dyed latexsuch as those described Snyder (EP 0 280 559 and 0 281 327) and dyesencapsulated in liposomes as described by Campbell et al. (U.S. Pat. No.4,703,017). Other direct labels that may be used will be apparent tothose of skill in the art.

[0247] Examples of suitable indirect labels include enzymes capable ofreacting with or interacting with a substrate to produce a detectablesignal (such as those used in ELISA and EMIT immunoassays), ligandscapable of binding a labeled moiety, and the like. Suitable enzymesuseful as indirect labels include, by way of example and not limitation,alkaline phosphatase, horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. Theuse of these enzymes in ELISA and EMIT immunoassays is described indetail in Engvall, 1980, Methods in Enzymology, 70:419-439 and U.S. Pat.No. 4,857,453.

[0248] Methods of labeling proteins and compounds with a variety oflabels such as those described above are well-known. Any of thesemethods may be used to label CLIC1s and/or candidate compounds. Forexample, a CLIC1 may be labeled with a fluorophore such as fluoresceinby incubating the CLIC1 with, for example, fluorescein isothiocyanate,using conventional techniques. Alternatively, a CLIC1 (or a candidatecompound produced by recombinant techniques) can be labeledmetabolically by culturing cells that express the CLIC1 in the presenceof culture medium supplemented with a metabolic label, such as, by wayof example and not limitation, [³⁵S]-methionine, one or more[¹⁴C]-labeled amino acids, one or more [¹⁵N]-labeled amino acids and/or[³H]-labeled amino acids (with the tritium substituted at non-labilepositions).

[0249] In one embodiment of the invention, candidate compounds may bescreened for the ability to bind a CLIC1 using an affinitychromatography technique. For example, a CLIC1 may be attached to achromatography resin according to standard techniques to create a CLIC1affinity resin and this CLIC1 affinity resin used to identify compoundsthat bind the resin. Alternatively, the candidate compound could bebound to the resin and the resin used to determine whether it binds aCLIC1. In another alternative embodiment, an active DL03 compound of theinvention may by attached to the chromatography resin. This DL03affinity resin may then be used to bind a CLIC1 and the bound complexused to identify compounds that compete for binding the CLIC1 with theactive DL03 compound, typically by washing the resin with a candidatecompound and determining whether the candidate compound disrupts theCLIC1-DL03 compound complex by assaying for the release of CLIC1 fromthe resin.

[0250] Although candidate compounds may be screened for the ability tobind a CLIC1 on a compound-by-compound basis, it may be more convenientto screen large numbers of candidate compounds simultaneously using oneof the many library screening methodologies known in the art. Oneart-known approach uses recombinant bacteriophage to produce largelibraries of peptides which can then be screened in a variety of formatsfor binding to a CLIC1. Using such phage methods, very large librariesof candidate peptides can be constructed (e.g., 10⁶-10⁸ peptides) andscreened for binding with a CLIC1. Methods for constructing andscreening such “phage display” libraries are described, for example, inScott & Smith, 1990, Science 249:386-390; Cwirla et al., 1990, Proc.Natl. Acad. Sci. 87:6378-6382; 1990); Devlin et al., 1990, Science249:404-406 (1990); U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,432,018;U.S. Pat. No. 5,580,717 and U.S. Pat. No. 5,723,286, the disclosures ofwhich are incorporated herein by reference. Other non-limiting examplesof recombinant library methodologies that may be used in connection withthe assays of the invention are described in U.S. Pat. No. 6,156,571;U.S. Pat. No. 6,107,059 and U.S. Pat. No. 5,733,731, the disclosures ofwhich are incorporated herein by reference.

[0251] A second art-known approach uses chemical methods to synthesizelibraries of compounds, such as small organic compounds, peptides and/orpeptide analogs, attached to beads or wafers that can then beconveniently screened for binding with a CLIC1. The libraries may beencoded or non-encoded. Methods of synthesizing such immobilizedlibraries, as well as methods of screening the libraries are described,for example, in Houghten, 1985, Proc. Natl. Acad. Sci. USA 82:5131-5735;Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al.,1987, J. Immunologic Method 102:259-274; Frank & Doring, 1988,Tetrahedron 44:6031-6040; Fodor et al., 1991, Science 251:767-773; Furkaet al., 1988, 4th International Congress of Biochemistry, Volume 5,Abstract FR:013; Furka, 1991, Int. J. Peptide Protein Res. 37:487-493;Frank, 1992, Tetrahedron 48:9217-9232; Needels et al., 1993, Proc. Natl.Acad. Sci. USA 90:10700-10704; DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909-6913; Frank et al., 1993, Biorg. Med. Chem. Lett.3:425-430; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926; WO 92/00252; WO 9428028; U.S. Pat. No. 6,329,143; U.S.Pat. No. 6291,183; U.S. Pat. No. 5,885,837; U.S. Pat. No. 5,424,186;U.S. Pat. No. 5,384,261; U.S. Pat. No. 6,165,717; U.S. Pat. No.6,143,497; U.S. Pat. No. 6,140,493; U.S. Pat. No. 5,789,162; U.S. Pat.No. 5,770,358; U.S. Pat. No. 5,708,153; U.S. Pat. No. 5,639,603; U.S.Pat. No. 5,541,061; U.S. Pat. No. 5,525,735; U.S. Pat. No. 5,525,734;U.S. Pat. No. 6,261,776; U.S. Pat. No. 6,239,273; U.S. Pat. No.5,846,839; U.S. Pat. No. 5,770,455; U.S. Pat. No. 5,770,157; U.S. Pat.No. 5,609,826; U.S. Pat. No. 6,001,579; U.S. Pat. No. 5,968,736; U.S.Pat. No. 5,962,337; U.S. Pat. No. 5,789,172; U.S. Pat. No. 5,721,099;U.S. Pat. No. 5,565,324; U.S. Pat. No. 5,010,175; and U.S. Pat. No.4,631,211, the disclosures of which are incorporated herein byreference. For reviews of some of these techniques, see Ellman et al.,1996, Account, Chem. Res. 29:132-143; Gallop et al., 1994, J. Med. Chem.37:1233-1251; Gordon et al., 1994, J. Med. Chem. 37:1385-1401.Non-limiting examples of solid-phase chemical synthesis strategies andconditions useful for synthesizing combinatorial libraries of smallorganic and other compounds may be found in Bunin, 1998, TheCombinatorial Index, Academic Press, London, England (see, e.g., Chapter1-5) and Hermkens et al., 1996, Tetrahedron 52:4527-4554, as well as thereferences cited therein, the disclosures of which are incorporatedherein by reference.

[0252] Another art-known approach utilizes solution-phase chemicalsynthesis techniques to synthesize libraries of compounds, such as, forexample, libraries of small organic compounds, which may then bescreened in the assays of the invention. Methods for synthesizing andscreening such solution-phase libraries are well-known and aredescribed, for example, in Bunin, 1998, The Combinatorial Index,Academic Press, England (see, e.g., Chapter 6); WO 95/02566; U.S. Pat.No. 5,962,736; U.S. Pat. No. 5,766,481; U.S. Pat. No. 5,736,412 and U.S.Pat. No. 5,712,171, and the references cited therein; the disclosures ofwhich are incorporated herein by reference. Additional review articles,references, patents and books describing myriad techniques forsynthesizing and screening libraries of compounds for the ability tobind another compound such as a CLIC1 can be found at Lebl & Leblova:Dynamic Database of References in Molecular Diversity, Internethttp://www.5z.com (see especially the diversity information pages athttp://www.5z.com/divinfo).

[0253] Once a candidate compound that binds the CLIC1 has beenidentified, further assays may be carried out to characterize thebinding characteristics of the compound, for example, to determine itsbinding affinity, dissociation constant (Kd), on- and/or off-rates,etc., using well-known techniques. For example, binding affinities canbe determined using saturation kinetics and Scatchard analysis. Forsaturation kinetics, the binding assay can be performed with increasingconcentrations of the candidate compound, which is typically labeledwith, for example, a radiolabel. Competitive binding experiments with anactive DL03 or other active compounds, for example peptide DL03wt (SEQID NO: 1), peptide DL03IL (SEQ ID NO: 2), peptide DL03KP (SEQ ID NO: 3),or peptide DL03LA (SEQ ID NO: 4), can be carried out with increasingconcentrations of unlabeled candidate compound and a fixed concentrationof labeled (for example radiolabled) active DL03 or other compounds.

[0254] An alternative method for characterizing receptor/ligand bindingcharacteristics of a plurality of compounds in parallel that may beadapted for use in connection with the invention is described in U.S.Pat. No. 5,324,633.

[0255] In one embodiment of the invention, the candidate compoundsidentified will have a dissociation constant (Kd) for CLIC1 on the orderof 1 mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or even lower. Inanother embodiment of the invention, the candidate compounds identifiedwill exhibit an IC₅₀ in a competitive binding assay with an active DL03compound of the invention or another compound that competes for bindinga CLIC1 with an active DL03 compound of the invention on the order of 1mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 μM or even lower. In thiscontext, the IC₅₀ represents the concentration of candidate compoundthat displaces 50% of the bound DL03 or other compound. Suitable assaysfor measuring such IC₅₀s are well-known.

[0256] If desired, the ability of identified candidate compounds tomodulate or regulate IL-4 induced IgE production and/or processesassociated therewith can be confirmed in in vitro assays, such as thosedescribed herein in connection with the identification of the DL03compoundsfor example as described in the Examples. Other assays that arewell known in the art may be used, for example, those described in U.S.Pat. No. 5,958,707.

[0257] Knowledge of the interaction surface between a DL03 compound ofthe invention such as peptide DL03wt (SEQ ID NO: 1), peptide DL03IL (SEQID NO: 2), peptide DL03KP (SEQ ID NO: 3) and/or DL03LA (SEQ ID NO: 4)and a CLIC1, and in particular the CLIC1 amino acids involved in bindingthe DL03 compound, can also provide useful information for identifyingcompounds that bind a CLIC1. Identification and screening of CLIC1binding compounds is further facilitated by determining structuralfeatures of a CLIC1-DL03 compound complex, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry or any other techniques for structure determination. Thesetechniques provide for the rational design or in silico identificationof compounds that bind a CLIC1. The crystal structure of a Soluble formof hCLIC1 has been determined to 1.4-Å resolution. (Harrop et al.(2001), supra; PDB accession code 1KOM)

[0258] Candidate compounds identified using the screening assays of theinvention may be agonists or antagonists of the CLIC1. Thus, theidentified compounds may bind to the CLIC1 without activating it or mayinhibit one or more biological activities of CLIC1 (antagonist) or,alternatively, the identified compounds may activate one or morebiological activities of the CLIC1 (agonists). These functional assaysalso provide an indirect means of assessing whether a candidate compoundbinds a CLIC1. Observation of agonist or antagonist activity indicatesthe candidate compound binds the CLIC1. Thus, the method of theinvention may employ functional assays of CLIC1 activity to determinewhether a candidate compound binds CLIC1. Suitable methods fordetermining the effect of the candidate compound on the activity ofCLIC1 are well known and include, for example, methods described inHarrop et al. (2000, supra), Tulk et al. (2002, supra), Tulk et al.(2000, supra), Tonini et al. (2000, supra), Valenzuela et al. (2000,supra) and U.S. Pat No. 5,854,411.

[0259] Cell-based functional assays for CLIC1 targets may be carried outin living cells that express CLIC, either endogenously or recombinantly.For example, suitable methods include electrophysiological and membranepotential assays such as patch clamping, fast and slow response dyes,fluorescence resonance energy transfer (FRET), ion tracer assays, etc.).For reviews of these techniques, see Mattheakis et al., Curr Opin DrugDiscov Devel. 2001; Gonzalez et al., Drug Discov Today. September1999;4(9):431-439. January;4(1):124-34., Xu et al., Drug Discov Today.Dec. 15, 2001;6(24):1278-1287, as well as the references cited therein,the disclosures of which are incorporated herein by reference.

[0260] Kits

[0261] The invention also provides kits for carrying out the variousscreening assays and methods of the invention. Such kits will typicallyinclude a CLIC1 and a compound that competes for binding with the CLIC1,such as an active DL03 compound. The CLIC1 and/or compound may belabeled or unlabeled. The kit may further include additional componentsuseful for carrying out the assays and methods. Non-limiting examples ofsuch additional components include labels, labeling reagents, bindingbuffers, etc. The kit may also include instructions teaching its methodsof use. In one embodiment, the kit comprises and CLIC1 and a compoundselected from peptide DL03wt (SEQ ID NO: 1), peptide DL03IL (SEQ, ID NO:2), peptide DL03KP (SEQ ID NO: 3), peptide DL03LA (SEQ ID NO: 4) peptideDL03TS (SEQ ID NO: 5), peptide DL03GA (SEQ ID NO: 6), peptide DL03TT(SEQ ID NO: 7), peptide DL03AS (SEQ ID NO: 8), peptide DL03MT (SEQ IDNO: 9) and an analog thereof.

[0262] Uses of the DL03 Compounds and Identified Compounds

[0263] As discussed previously, the active DL03 compounds of theinvention and/or the active CLIC1-binding compounds identified by theabove-described screening methods (referred to collectively as “activecompounds”), can be used in a variety of in vitro, in vivo and ex vivoapplications to regulate or modulate processes involved with theproduction and/or accumulation of IgE. For example, the active compoundscan be used to modulate, and in particular inhibit, any or all of thefollowing processes in vitro, in vivo or ex vivo: IgE production and/oraccumulation; the IL-4 receptor-mediated signaling cascade leading toisotype switching and/or production of IgE; IL-4 induced switching ofB-cells to produce IgE, IL-4 mediated IgE production; and IL-4 inducedgermline ε transcription. In a specific embodiment of the invention, theactive compounds may be used to treat or prevent diseases characterizedby, caused by or associated with production and/or accumulation of IgE.Such treatments may be administered to animals in veterinary contexts orto humans. Diseases that are characterized by, caused by or associatedwith IgE production and/or accumulation, and that can therefore betreated or prevented with the active compounds include, by way ofexample and not limitation, anaphylactic hypersensitivity or allergicreactions and/or symptoms associated with such reactions, allergicrhinitis, allergic conjunctivitis, systemic mastocytosis, hyper IgEsyndrome, IgE gammopathies, atopic disorders such as atopic dermatitis,atopic eczema and/or atopic asthma, and B-cell lymphoma.

[0264] When used to treat or prevent such diseases, the active compoundsmay be administered singly, as mixtures of one or more active compoundsor in mixture or combination with other agents useful for treating suchdiseases and/or symptoms associated with such diseases. The activecompounds may also be administered in mixture or in combination withagents useful to treat other disorders or maladies, such as steroids,membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptorinhibitors, IgE receptor inhibitors, β-agonists, tryptase inhibitors andantihistamines, to name a few. The active compounds may be administeredper se or as pharmaceutical compositions.

[0265] Pharmaceutical compositions comprising the active compounds ofthe invention may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping or lyophilization processes. The compositionsmay be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically. The actual pharmaceuticalcomposition administered will depend upon the mode of administration.Virtually any mode of administration may be used, including, for exampletopical, oral, systemic, inhalation, injection, transdermal, etc.

[0266] The active compound may be formulated in the pharmaceuticalcompositions per se, or in the form of a pharmaceutically acceptablesalt. As used herein, the expression “pharmaceutically acceptable salt”means those salts which retain substantially the biologicaleffectiveness and properties of the active compound and which is notbiologically or otherwise undesirable. Such salts may be prepared frominorganic and organic acids and bases, as is well-known in the art.Typically, such salts are more soluble in aqueous solutions than thecorresponding free acids and bases.

[0267] For topical administration, the active compound(s) may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

[0268] Systemic formulations include those designed for administrationby injection, e.g., subcutaneous, intravenous, intramuscular,intrathecal or intraperitoneal injection, as well as those designed fortransdermal, transmucosal oral or pulmonary administration.

[0269] Useful injectable preparations include sterile suspensions,solutions or emulsions of the active compound(s) in aqueous or oilyvehicles. The compositions may also contain formulating agents, such assuspending, stabilizing and/or dispersing agent. The formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmultidose containers, and may contain added preservatives.

[0270] Alternatively, the injectable formulation may be provided inpowder form for reconstitution with a suitable vehicle, including butnot limited to sterile pyrogen free water, buffer, dextrose solution,etc., before use. To this end, the active compound(s) may dried by anyart-known technique, such as lyophilization, and reconstituted prior touse.

[0271] For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants areknown in the art.

[0272] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art with, forexample, sugars or enteric coatings.

[0273] Liquid preparations for oral administration may take the form of,for example, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

[0274] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0275] For rectal and vaginal routes of administration, the activecompound(s) may be formulated as solutions (for retention enemas)suppositories or ointments containing conventional suppository basessuch as cocoa butter or other glycerides.

[0276] For administration by inhalation, the active compound(s) can beconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0277] For prolonged delivery, the active compound(s) can be formulatedas a depot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt.

[0278] Alternatively, transdermal delivery systems manufactured as anadhesive disc or patch which slowly releases the active compound(s) forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activecompound(s). Suitable transdermal patches are described in for example,U.S. Pat. No. 5,407,713.; U.S. Pat. No. 5,352,456; U.S. Pat. No.5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat.No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S.Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110;and U.S. Pat. No. 4,921,475.

[0279] Alternatively, other pharmaceutical delivery systems may beemployed. Liposomes and emulsions are well-known examples of deliveryvehicles that may be used to deliver active compounds(s). Certainorganic solvents such as dimethylsulfoxide (DMSO) may also be employed,although usually at the cost of greater toxicity.

[0280] The pharmaceutical compositions may, if desired, be presented ina pack or dispenser device which may contain one or more unit dosageforms containing the active compound(s). The pack may, for example,comprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.

[0281] Gene Therapy

[0282] As will be recognized by skilled artisans, active compound(s)that are peptides composed wholly of genetically-encoded amino acids maybe administered utilizing well-known gene therapy techniques. Accordingto such techniques, a gene encoding the active compound may beintroduced either in vivo, ex vivo, or in vitro in a viral vector. Suchvectors include an attenuated or defective DNA virus, such as but notlimited to, herpes simplex virus (HSV), papillomavirus, Epstein Barrvirus (EBV), adenovirus, adeno-associated virus (AAV), and the like.Defective viruses, which entirely or almost entirely lack viral genes,are preferred.

[0283] Defective virus is not infective after introduction into a cell.Use of defective viral vectors allows for administration to cells in aspecific, localized area, without concern that the vector can infectother cells. For example, in the treatment of the various diseasesdescribed herein, lymphocyte B-cells can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus I (HSV1) vector (Kaplitt et al., 1991, Molec.Cell. Neurosci. 2:320-330), an attenuated adenovirus vector, such as thevector described by Stratford-Perricaudet et al., 1992, J. Clin. Invest.90:626-630 and a defective adeno-associated virus vector (Samulski etal., 1987, J. Virol. 61:3096-3101; Samulski et al., 1989, J. Virol.63:3822-3828).

[0284] Preferably, for in vitro administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors (see, e.g., Wilson, 1995, Nat.Med. 1(9):887-889). In addition, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

[0285] In another embodiment the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al, U.S. Pat. No. 5,399,346;Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol.62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; Dougherty et al.,WO 95/07358; and Kuo et al., 1993, Blood 82:845. Targeted gene deliveryis described in WO 95/28494.

[0286] Alternatively, the vector can be introduced by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417; Mackey et al.,1988, Proc. Natl. Acad. Sci. USA 85:8027-8031). The use of cationiclipids may promote encapsulation of negatively charged nucleic acids,and also promote fusion with negatively charged cell membranes (Felgner& Ringold, 1989, Science 337:387-388). The use of lipofection tointroduce exogenous genes into the specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (see Mackey et al., 1988, supra). Targetedpeptides, e.g., hormones or neurotransmitters, and proteins such asantibodies, or non-peptide molecules could be coupled to liposomeschemically.

[0287] It is also possible to introduce the vector as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu & Wu, 1988, J. Biol. Chem., 263:14621-14624; CanadianPatent Application No.2,012,311).

[0288] Naked nucleic acids encoding the active compound may also beintroduced using the gene-activated matrices described, for example, inU.S. Pat. No. 5,962,427.

[0289] Effective Dosages

[0290] The active compound(s) of the invention, or compositions thereof,will generally be used in an amount effective to treat or prevent theparticular disease being treated. The compound(s) may be administeredtherapeutically to achieve therapeutic benefit or prophylactically toachieve prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated,e.g., eradication or amelioration of the underlying allergy, atopicdermatitis, atopic eczema or atopic asthma, and/or eradication oramelioration of one or more of the symptoms associated with theunderlying disorder such that the patient reports an improvement infeeling or condition, notwithstanding that the patient may still beafflicted with the underlying disorder. For example, administration ofan active compound to a patient suffering from an allergy providestherapeutic benefit not only when the underlying allergic response iseradicated or ameliorated, but also when the patient reports a decreasein the severity or duration of the symptoms associated with the allergyfollowing exposure to the allergen. Therapeutic benefit also includeshalting or slowing the progression of the disease, regardless of whetherimprovement is realized.

[0291] For prophylactic administration, the active compound may beadministered to a patient at risk of developing a disorder characterizedby, caused by or associated with IgE production and/or accumulation,such as the various disorders previously described. For example, if itis unknown whether a patient is allergic to a particular drug, theactive compound may be administered prior to administration of the drugto avoid or ameliorate an allergic response to the drug. Alternatively,prophylactic administration may be applied to avoid the onset ofsymptoms in a patient diagnosed with the underlying disorder. Forexample, an active compound may be administered to an allergy suffererprior to expected exposure to the allergen. Active compounds may also beadministered prophylactically to healthy individuals who are repeatedlyexposed to agents known to induce an IgE-related malady to prevent theonset of the disorder. For example, an active compound may beadministered to a healthy individual who is repeatedly exposed to anallergen known to induce allergies, such as latex allergy, in an effortto prevent the individual from developing an allergy.

[0292] The amount of active compound(s) administered will depend upon avariety of factors, including, for example, the particular indicationbeing treated, the mode of administration, whether the desired benefitis prophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular active compound, etc. Determination of an effectivedosage is well within the capabilities of those skilled in the art.

[0293] Initial dosages may be estimated initially from in vitro assays.For example, an initial dosage for use in animals may be formulated toachieve a circulating blood or serum concentration of active compoundthat inhibits about 25% or more of IL-4 induced IgE production, or aprocess associated therewith, such as germline ε transcription, asmeasured in an in vitro assay. Alternatively, an initial dosage for usein animals may be formulated to achieve a circulating blood or serumconcentration of active compound that is equal to or greater than theIC₅₀ as measured in a CLIC1 competitive binding assay with an activeDL03 compound of the invention, such as paptideDL03wt (SEQ ID NO: 1),peptide DL03IL (SEQ ID NO: 2), peptide DL03KP (SEQ ID NO: 3), or peptideDL03LA (SEQ ID NO: 4). Calculating dosages to achieve such circulatingblood or serum concentrations taking into account the bioavailability ofthe particular active compound is well within the capabilities ofskilled artisans. For guidance, the reader is referred to Fingl &Woodbury, “General Principles,” In: The Pharmaceutical Basis ofTherapeutics, Chapter 1, pp. 1-46, 1975, and the references citedtherein.

[0294] Initial dosages can also be estimated from in vivo data, such asanimal models. Animals models useful for testing the efficacy ofcompounds to treat or prevent diseases characterized by, caused by orassociated with IgE production and/or accumulation are well-known in theart. Suitable animal models of hypersensitivity or allergic reactionsare described in Foster, 1995, Allergy 50(21Suppl):6-9, discussion 34-38and Tumas et al., 2001, J. Allergy Clin. Immunol. 107(6): 1025-1033.Suitable animal models of allergic rhinitis are described in Szelenyi etal., 2000, Arzneimittelforschung 50(11):1037-42; Kawaguchi et al., 1994,Clin. Exp. Allergy 24(3):238-244 and Sugimoto et al., 2000,Immunopharmacology 48(1):1-7. Suitable animal models of allergicconjunctivitis are described in Carreras et al., 1993, Br. J.Ophthalmol. 77(8):509-514; Saiga et al., 1992, Ophthalmic Res.24(l):45-50; and Kunert et al., 2001, Invest. Ophthalmol. Vis. Sci.42(11):2483-2489. Suitable animal modules of systemic mastocytosis aredescribed in O'Keefe et al., 1987, J. Vet. Intern. Med. 1(2):75-80 andBean-Knudsen et al., 1989, Vet. Pathol. 26(1):90-92. Suitable animalmodels of hyper IgE syndrome are described in Claman et al., 1990, Clin.Immunol. Immunopathol. 56(1):46-53. Suitable animal models of B-celllymphoma are described in Hough et al., 1998, Proc. Natl. Acad. Sci. USA95:13853-13858 and Hakim et al., 1996, J. Immunol. 157(12):5503-5511.Suitable animal models of atopic disorders such as atopic dermatitis,atopic eczema and atopic asthma are described in Chan et al., 2001, J.Invest. Dermatol. 117(4):977-983 and Suto et al., 1999, Int. Arch.Allergy Immunol. 120(Suppl 1):70-75. Ordinarily skilled artisans canroutinely adapt such information to determine dosages suitable for humanadministration.

[0295] Dosage amounts will typically be in the range of from about 1mg/kg/day to about 100 mg/kg/day, 200 mg/kg/day, 300 mg/kg/day, 400mg/kg/day or 500 mg/kg/day, but may be higher or lower, depending upon,among other factors, the activity of the active compound, itsbioavailability, the mode of administration and various factorsdiscussed above. Dosage amount and interval may be adjusted individuallyto provide plasma levels of the active compound(s) which are sufficientto maintain therapeutic or prophylactic effect. In cases of localadministration or selective uptake, such as local topicaladministration, the effective local concentration of active compound(s)may not be related to plasma concentration. Skilled artisans will beable to optimize effective local dosages without undue experimentation.

[0296] The compound(s) may be administered once per day, a few orseveral times per day, or even multiple times per day, depending upon,among other things, the indication being treated and the judgement ofthe prescribing physician.

[0297] Preferably, the active compound(s) will provide therapeutic orprophylactic benefit without causing substantial toxicity. Toxicity ofthe active compound(s) may be determined using standard pharmaceuticalprocedures. The dose ratio between toxic and therapeutic (orprophylactic) effect is the therapeutic index. Active compound(s) thatexhibit high therapeutic indices are preferred.

[0298] The invention having been described, the following examples areoffered by way of illustration and not limitation.

EXAMPLES

[0299] Identification of Peptide DL03wt from a Random Library of Peptide20-mers

[0300] Peptide DL03wt (SEQ ID NO: 1) was identified by screening aretroviral library of random peptide 20-mers for the ability to inhibitIL-4 induced germline ε transcription using the HBEGF2a/diphtheria dualreporter phenotypic screening system described in WO 01/31232. Toconstruct the random library, A5T4 reporter cells (described in moredetail below) were infected with an infectious retroviral library ofrandom peptide 20-mers (prepared as described in WO 97/27213; see alsoWO 01/34806 at page 39, line 36 through page 40, line 19). Theretroviral vector used includes a gene encoding blue fluorescent protein(BFP) fused upstream of the region encoding the random peptide via alinker region encoding an α-helical peptide linker (expression fusionproduct is referred to as “BFP-peptide”). Expression of the BFP-peptideproduct is controlled by a promoter sensitive to thetetracycline-regulated transactivator such that expression of theBFP-peptide is regulated by tetracycline (Tet) or doxycycline (Dox). SeeU.S. patent application Ser. No. 10/096,339, entitled “Methods andCompositions for Screening for Altered Cellular Phenotypes”, filed onMar. 8, 2002. The BFP reporter gene provides a rapid phenotypic assay todetermine whether cells were infected: infected cells expressBFP-peptide and fluoresce blue (phenotype BFP⁺), uninfected cells do notexpress BFP-peptide, and do not fluoresce blue (phenotype BFP⁻). Toreduce the number of stop codons, the region of the vector encoding therandom peptide was of the sequence (NNK)₂₀, where each N independentlyrepresents A, T, C or G and K represents T or G. The library was alsobiased to account for degeneracy in the genetic code.

[0301] The A5T4 reporter cell line was engineered from BJAB B-cells(Menezes et al., 1975, Biomedicine 22:276-284; Source: Yoshinobu Matsuo,PhD., Fujisaki Cell Center, Hayashibara Biochemical Labs, Inc., 675-1Fujisaki, Okayama 702-8006, Japan) and includes a reporter gene encodingthe HBEGF2a/GFP dual function reporter positioned downstream of anengineered 600 base pair IL-4 responsive fragment of an ε promoter (FIG.1; see also WO 99/58663) such that ultimate expression of the dualfunction reporter is driven by the ε promoter. When expressed, the dualfunction reporter cleaves into two pieces, a heparin-binding epidermalgrowth factor-like growth factor (HBEGF) and a green fluorescent protein(GFP), via the self-cleaving 2a sequence (Donnelly et al., 2001, J. Gen.Viol. 82:1027-1041; Donnelly et al., 1997, J. Gen. Virol. 78:13-21;Mattion et al., 1996, J. Virol. 70:8124; Ryan et al., 1994, EMBO J13:928-33 Ryan et al., 1991, J. Gen. Virol. 72:2727; Hellen et aL, 1989,Biochem. 28:9881; see also, WO 99/58663). In this reporter system, cellsectopically expressing HBEGF are capable of translocating diphtheriatoxin (DT) into their cytoplasm, leading to rapid, acute cytotoxicity.Cells that do not express HBEGF are spared this fate and continue tosurvive even in the presence of high concentrations of DT. The A5T4reporter cell line was further engineered to express thetetracycline-regulated transactivator (tTA), allowing for regulation ofpeptide library expression with tetracycline (Tet) or doxycycline (Dox).See U.S. patent application Ser. No. 10/096,339, the disclosure of whichis incorporated herein by reference. Thus, according to this dualphenotypic reporter system, unstimulated control cells expressing arandom peptide fluoresce blue (BFP⁺) in the absence of Tet or Dox. Inthe presence of Tet or Dox, the peptide is not made and the cells areBFP⁻. Following stimulation with IL-4, BFP⁺ cells expressing anon-inhibitory peptide fluoresce green and, in addition, are sensitiveto DT. Stimulated BFP⁺ cells expressing an inhibitory peptide do notfluoresce green and are not DT sensitive. The toxin-conditionalselection and Tet or Dox-controlled peptide expression features of theA5T4 screening line are illustrated in FIGS. 2A & 2B, respectively.

[0302] Following infection, the library was enriched for cellsexpressing peptides that inhibit IL-4 induced ε transcription asgenerally outlined in the top half of FIG.3 and sorted by FACS intosingle cell clones. The clones were then screened as generallyillustrated in the lower half of FIG. 3. Briefly, for screening, eachclone was divided into two populations and one population was treatedwith Dox (10 ng/ml). After 5 days, both populations were stimulated withIL-4 (final conc. 60 U/mL; PeptroTech, Inc.) and, after 3 more days,both populations were analyzed by FACS to measure BFP and GFPfluorescence. FACS data were converted to a “reporter ratio”, which isdefined for this purpose as the ratio of the geometric mean of GFPfluorescence of the +IL-4/+Dox to the +IL-4/−Dox populations. Cellsexpressing a peptide that inhibits gernline ε transcription have areporter ratio of≧1.1. A repoiter ratio of≧1.2 is indicative of stronginhibition.

[0303] The sequences of peptides expressed by positive clones (reporterratios of ≧1.1) were obtained by RT-PCR amplification of the integratedpeptide-expressing sequences. In this experiment, of 2.4×10⁹ A5T4 cellsinfected, 218 positive clones were identified, 199 of which were unique.From this same experiment, 155 total clones with a reporter ratio of≧1.19 were identified, 136 of which were unique. Clone DL03, whichencodes peptide DL03wt, was amongst the positive clones identified(clone DL03 had a reporter ratio of 1.32).

[0304] Clone DL03 Transfers its Phenotype Into Naive Cells

[0305] The ability of peptide DL03wt (SEQ ID NO: 1) to inhibit germlineε transcription was confirmed in naïve cells. Briefly, Phoenix cellswere transfected with a retroviral vector encoding a BFP-DL03wt (SEQ IDNO: 1) peptide fusion as described in WO 99/58663 and WO 97/27213. NaïveA5T4 cells were infected with the resultant virions and grown for 3days.

[0306] The infected cells were stimulated with IL-4 (60 U/mL) and, after3 days, the cells were assessed by FACS for BFP and GFP. The FACS dataare presented in FIG. 4. As illustrated in FIG. 4, there are twopopulations of cells: infected cells that express the BFP-peptide fusion(BFP⁺) and uninfected cells that do not (BFP⁻). The BFP fluorescencedata corresponding to the BFP⁺ and BFP⁻ populations are provided inPanel A. The GFP fluorescence data corresponding to the BFP⁺ and BFP⁻populations are presented in Panel B. The reporter ratio for thispurpose is determined as the geometric mean of the GFP fluorescence ofthe BFP⁻ population divided by the geometric mean of the GFPfluorescence of the BFP⁺ population as presented in panel C. Thereporter ratio for the DL03 in this re-infection assay was 1.50.

[0307] Peptide DL03wt Inhibits Transcription of an Endogenous Germline εPromoter

[0308] The ability of peptide DL03wt (SEQ ID NO: 1) to inhibittranscription of an endogenous germline ε promoter was confirmed using aTAQMAN® assay (Roche Molecular, Alameda, Calif.). Briefly, A5T4 cellswere infected with retrovirus capable of expressing peptide DL03wt (SEQID NO: 1) (prepared as described above). The cells were sorted for BFP⁺to select for infected cells. Infected cells were divided into twopopulations. One population was exposed to Dox (10 ng/ml). Bothpopulations were stimulated with IL-4 (60 U/ml). After 3 days, the cellswere pelleted and the pellets assayed for ε promoter transcription usinga TAQMAN assay performed as described in Applied Biosystems Protocol4310299 (available at http://www.appliedbiosystems.com). The primers andprobe, which are specific for the transcription product driven by theA5T4 endogenous & promoter, were as follows (the probe was labeled atthe 5′-end with Fam and at the 3′-end with Tamra): ε forward primer:ATCCACAGGCACCAAATGGA (SEQ ID NO:12) ε reverse primer:GGAAGACGGATGGGCTCTG (SEQ ID NO:13) ε probe: ACCCGGCGCTTCAGCCTCCA (SEQ IDNO:14)

[0309] The measured endogenous ε inhibition ratio, defined as the ratioof the relative expression units (TAQMAN quantitative PCR of εtranscription product) of +IL-4/+Dox to +IL-4/−Dox cells, was 2.11(average of 3 values; p=0.0035), indicating that peptide DL03wt (SEQ IDNO: 1) strongly inhibits the endogenous germline ε promoter.

[0310] Peptide DL03wt is Selective for the Germline c Promoter

[0311] To demonstrate selectivity for the germline ε promoter, peptideDL03wt (SEQ ID NO: 1) was tested for inhibition of germline αtranscription. The assay was similar to that described in theimmediately preceding section, except that ST486 cells (ATCC # CRL-1647)engineered to express the tetracycline-regulated transactivator wereinfected and the infected cells were stimulated with TGF-β (40 ng/ml;Peprotech). The primers and probe, which are specific for thetranscription product driven by the ST486 endogenous α promoter, were asfollows (the probe was labeled at the 5′-end with Fam and at the 3′-endwith Tamra): α forward primer: CAGCACTGCGGGCCC (SEQ ID NO:15) α reverseprimer: TCAGCGGGAAGACCTTGG (SEQ ID NO:16) α probe:CCAGCAGCCTGACCAGCATCCC (SEQ ID NO:17)

[0312] The measured endogenous a inhibition ratio was 0.66 (average of 3values p=0.3228), indicating that peptide DL03wt (SEQ ID NO: 1) does notinhibit transcription of the germline α promoter. These data confirmthat peptide DL03wt (SEQ ID NO: 1) is a selective inhibitor of germlineε transcription.

[0313] IgE Synthesis and ELISA Assay

[0314] This example describes an IgE synthesis mixed lymphocyte andELISA assay that may be used to assess the amount of IgE produced bycells such as human peripheral blood lymphocytes or other lymphaticcells in the presence and absence of candidate compounds, such as DL03compounds and/or compounds identified in the screening assays of theinvention.

[0315] IgE Synthesis Assay

[0316] (a) Materials

[0317] In the various protocols that follow below, the followingmaterials are used:

[0318] Heparin (Sigma H3393, St. Louis, Mo.)

[0319] Histopaque 1077 tubes (Sigma A0561, St. Louis, Mo.)

[0320] Iscove's Modified Dulbeccos Medium (“IMDM); Sigma 13390, St.Louis, Mo.)

[0321] Bovine Serum Albumin (“BSA”, Sigma A9418, St. Louis, Mo.).

[0322] Fetal Bovine Serum (“FCS”; Sigma F7524, St. Louis, Mo.). (theserum is heat inactivated prior to use at 56° C. for 30 minutes,aliquoted and stored at −20° C.; “HI-FCS”)

[0323] Human Transferrin (Sigma T2252, St. Louis, Mo.)

[0324] Bovine Insulin (Sigma I1882, St. Louis, Mo.)

[0325] 200 mM L-Glutamine (“L-Gln”; Sigma G7513, St. Louis, Mo.) (storedas 5 ml aliquots at −20° C.)

[0326] Pen/Strep 10% solution (Sigma P0781, St. Louis, Mo.) (stored as 5ml aliquots at −20° C.)

[0327] PBS Dulbeccos (Gibco BRL 14190-094, now Invitrogen, Carlsbad,Calif.)

[0328] DMSO (Sigma D2650, St. Louis, Mo.)

[0329] Recombinant Human IL-4 (R&D Systems 204-1L, Minneapolis, Minn.)(stored as a stock solution of 100,000 U/ml in culture medium at −20°C.)

[0330] 96 well tissue culture plates (Costar 3595, Corning Inc., LifeSciences, Acton Mass.)

[0331] 96 well dilution blocks (Porvair 219008, Shepperton, UK)

[0332] Culture Medium: supplement 500 ml IMDM with 0.5% BSA (2.5 g), 10%HI-FCS (50 ml), 25 mg human transferrin, 2.5 mg bovine insulin, 2 mML-Gln (5 ml) and 1% pen/strep (5 mL). Filter sterilize before use.

[0333] (b) Blood Collection

[0334] Make up a 1 mg/ml solution of heparin in sterile PBS and place 1ml in each sterile 50 ml centrifuge tube required. Collect 50 ml ofvenous blood from a healthy human volunteer per centrifuge tube.

[0335] (c) Lymphocyte Isolation

[0336] Lymphocytes are isolated from the blood according to the protocolbelow:

[0337] 1. Dilute blood with an equal volume of PBS containing 2% HI-FCS.

[0338] 2. Add 20 ml diluted blood to each histopaque tube. Thehistopaque tubes should be warmed to room temperature before use. Theycan be left overnight at room temp in the dark and then spun at 1000 rpmfor 5 min to settle the contents before use. Spin at 1000 g for 35 minat room temperature in a benchtop centrifuge with the break set to off.

[0339] 3. Draw off the upper plasma layer and discard.

[0340] 4. Draw off the lymphocyte layer into a sterile centrifuge tube.If there is clear definition between the bottom of the lymphocyte layerand the top of the frit, remove only the lymphocyte layer, not all theliquid above the frit.

[0341] 5. Add 30 ml PBS-2% HI-FCS to each 10 ml of cell suspension andspin at 1000 rpm for 10 min at room temp.

[0342] 6. Discard the supernatant and resuspend each cell pellet in 5-10ml PBS-2%HI-FCS. Transfer the suspensions to a single tube and bring thevolume to 40 ml with PBS-2% HI-FCS. Spin at 1000 rpm for 10 min at roomtemp.

[0343] 7. Repeat Step 6.

[0344] 8. Wash the cells once with 40 ml culture medium.

[0345] 9. Discard the supernatant and resuspend the pellet in 10 ml orless culture medium.

[0346] 10. Count the cells (using a Neubauer haemocytometer, a CoulterMax-M cell counter or other counter).

[0347] 11. Resuspend the cells in culture medium to a concentration of2×10⁶ cells/ml. The cells can be left at this stage until they areneeded for assay set up.

[0348] (d) Assay Set Up

[0349] The assay is carried out as follows:

[0350] 1. Dissolve test compounds in DMSO to give a 10 mM stocksolution. If necessary, sonicate the stock solution to aid dissolutionof the compound.

[0351] 2. Dilute each compound stock solution 1:20 with culture mediumto yield a 500 μM solution in 5% DMSO. Serially dilute this 500 μM stocksolution 1:10 with culture medium several times to provide enough stocksolutions to test the compounds over a range of concentrations (e.g.,from 1 nM to 10 μM).

[0352] 3. Just prior to use, prepare a stock solution of IL-4 (1000 U/mlin culture medium).

[0353] 4. To test the compounds and prepare appropriate controls, add tothe appropriate wells of a multiwell plate the following reagents in thefollowing amounts: Reagent +IL-4 control −IL-4 control Sample TestCompound — —  50 μl IL-4  50 μl —  50 μl Culture Medium —  50 μl — 0.5%DMSO in  50 μl  50 μl — Culture Medium Cells 150 μl 150 μl 150 μl TotalVolume 250 μl 250 μl 250 μl

[0354] 5. Incubate the plates for 10-12 days at 37° C. in a CO₂incubator (5% CO₂/95% O₂). Following incubation, spin the plates at 1000rpm for 10 min and store at −20° C. until ready for the ELISA detectionassay that follows below.

[0355] ELISA Assay for Detection of IgE

[0356] (a) Materials

[0357] In the assay protocol that follows below, the following materialsare used:

[0358] Nunc maxisorp ELISA plates (GIBCO BRL, now Invitrogen, Carlsbad,Calif.)

[0359] Murine anti human IgE (GE1 clone) (Sigma, St. Louis, Mo.)

[0360] Phosphate buffered saline (“PBS”; Sigma. St. Louis, Mo.)

[0361] Bovine serum albumin (“BSA”; Sigma, St. Louis, Mo.)

[0362] Polyethylenesorbitan monolaurate (“TWEEN 20”; Sigma, St. Louis,Mo.)

[0363] OPD tablets (DAKO Corp., Carpenteria, Calif.)

[0364] Streptavidin biotinylated horseradish peroxidase complex(“Streptavidin HRP”; Amersham, Piscataway, N.J.)

[0365] Human Mycloma protein IgE (The Binding Site, Inc.; San Diego,Calif.)

[0366] Biotinylated anti human IgE (Vector Laboratories, Inc.,Burlingame, Calif.)

[0367] Hydrogen peroxide (Sigma, St. Louis, Mo.)

[0368] Distilled water (“dH₂O”)

[0369] Buffer A: 0.1 M NaHCO₃, pH 9.6

[0370] Buffer B: 0.1% TWEEN 20 in PBS

[0371] Buffer C: 1% BSA in Buffer B

[0372] Stop Solution: 0.6M H₂SO₄

[0373] (a) Protocol

[0374] The assay which measures the amount of IgE synthesized by thevarious controls and samples prepared above, is carried out as describedin the following protocol:

[0375] 1) Coat Nunc maxisorp plates with 50 μl of murine anti human IgE(1:2000 in Buffer A). Leave plates at 4° C. overnight. Plates can bestored in this way for a maximum of 7 days.

[0376] 2) Wash plates 3× with Buffer.

[0377] 3) Block any unbound sites on the plate by adding 200 μl ofbuffer C. Incubate the plates for at least 2 hours at room temperature.If blocking overnight incubate at 4 C. Blocked plates should only bekept for a maximum of 2 days.

[0378] 4) Wash plates 3× with Buffer B.

[0379] 5) Place 50 μl of the sample or standard IgE diluted in Buffer Cto each well. IgE standards are diluted to give a concentration range of100 ng/ ml-0 ng/ml. To make up the 100 ng/ml standard, carry out thefollowing dilutions of stock IgE (0.5 mg/ml) in buffer C: 1 in 50 togive a 10 μg/ml solution (a minimum of 10 μs must be transferred fromthe stock) followed by a 1 in 10 to givel 1 μg/ml and then a further 1in 10 to give 100 ng/ml. Double dilutions are then carried out in bufferC to give the rest of the standard curve. Carry out all dilutions inglass bottles (10 oz) and make up at least 1 ml of each so thatreasonable volumes are being transferred (one ELISA plate requires 100μls of each standard). For the pilot ELISA the standard curve is addedto each plate just after the samples have been added. Set up one ELISAplate with 3-4 columns of standards only. This will be used to determinedevelopment time at protocol Step 11.

[0380] For the pilot ELISA incubate the plates for one-two hours at roomtemperature

[0381] For the full ELISA incubate the plates overnight at 4° C.

[0382] 6) Wash plates 3× with Buffer B.

[0383] 7) Add 50 μl of biotinylated anti human IgE ({fraction (1/500)}dilution in Buffer B) to all wells. Incubate the plate for 1 hour atroom temperature.

[0384] 8) Wash plates 3× with Buffer B

[0385] 9) Add 50 μl of streptavidin HRP to each well ({fraction (1/800)}dilution in Buffer B). Incubate the plates for 45 mins to 1 hour at roomtemperature. The plates must not be left for longer than 1 hour at thisstage.

[0386] 10) Wash plates 3× with Buffer B.

[0387] 11) Add 50 μl of substrate (4 OPD tablets per 12 ml dH₂O plus 5μl hydrogen peroxide per 12 ml) to each well and wait for the color todevelop (this usually occurs within 10 minutes). Determine the timetaken to give the required OD (1.5-2 for 100 ng/ml of standard IgE)using a plate containing only a standard curve. Develop all test platesfor this amount of time and in batches of 5 plates. This is especiallyimportant if there are a large number of plates.

[0388] 12) Quench the reaction by adding 50 μl Stop Solution.

[0389] 13) Read plates at 492 nm within 30 minutes of stopping thereaction.

[0390] Peptide DL03wt Mediates its Inhibitory Action by Binding CLIC1

[0391] Identification of Potential Binding Partners for Peptide DL03wt

[0392] Potential binding partners for peptide DL03wt (SEQ ID: 1) wereidentified in a β-galactosidase yeast two-hybrid (YTH) assay usingpeptide DL03wt (SEQ ID: 1) as bait and a cDNA library constructed fromthe A5T4 reporter cell line as prey. Binding was assessed byβ-galactosidase quantification using BetaFluor (Novagen) as a substrate.A negative interaction control (no cDNA fused downstream of the GAL4activation domain sequence) was also run. A general outline of the YTHassay is illustrated in FIG. 5A. Following clustering, filtering toremove non-specific bait hits (e.g., GFP and BFP), singletons andclusters recognized by 10 or more cDNA baits (based upon historical YTHassays), and prioritization, 22 prey clones were identified as hits.

[0393] Potential targets identified in the YTH assay were reconfirmed asgenerally outlined in FIG. 6. The cDNA clones identified as hits in theYTH assay were purified and rescreened for interaction with the DL03wt(SEQ ID NO: 1) peptide in a YTH assay. The hit clones were also screenedin a control assay for interaction with the BFP using a vector (pGBKT7)that contained only the BFP and not the DL03 peptide. At this stage, 12putative target clones remained.

[0394] In order to further discriminate among the putative targets,functional/interaction profile analyses were carried out as described incopending application Ser. No. 10/095,659, entitled Methods ofIdentifying Protein Targets, filed Mar. 8, 2002.

[0395] Confirmation that Peptide DL03wt Mediates its Inhibitory Actionby Binding CLIC1

[0396] Identification of colony 3.2DL03_Y_AS_U_(—)101 as the bindingpartner for peptide DL03wt (SEQ ID NO: 1) was confirmed using theprofiling method described in copending application Ser. No. 10/095,659,entitled Methods of Identifying Protein Targets, filed Mar. 8, 2002 thedisclosure of which is incorporated herein by reference. The functionalprofile was obtained using the A5T4 reporter cell line and theinteraction profiles were obtained using the YTH assay and compared forcorrespondence. The main concept underlying this profiling method isthat mutants will tend to act the same way in both the functional assayand an interaction assay with the target polypeptide of the wild-typepeptide. That is, a mutant that exhibits an increase in function (ascompared to the wild-type peptide) in the functional assay will exhibitan increase in interaction (as compared to the wild-type peptide) in aYTH assay with the target polypeptide of the wild-type peptide. Statedanother way, the target polypeptide will yield an interaction profilethat corresponds closely to the functional profile when comparedvisually or by other means.

[0397] A collection of DL03 mutants was generated by replacing doubleamino acid residues in the sequence of DL03wt (SEQ ID NO: 1) with adifferent amino acid, typically an alanine, as described in copendingapplication Ser. No. 10/095,659. The functional profiles for the mutantsderived from DL03wt (SEQ ID NO: 1) was obtained by constructing andscreening for activity in the A5T4 reporter cell line in the mannerdescribed above. The activity of each mutant at the germline ε promoteris reflected in the reporter ratio as described above. The reporterratios were the functional values that were used to develop thefunctional profiles.

[0398] TABLE 2 depicts the amino acid sequences (the dual mutations areunderlined) of the mutants tested and the measured reporter ratios whenscreening in the A5T4 reporter line. TABLE 2 Re- Peptide porter NamePeptide Sequence Ratio SEQ ID NO DL03wt LYTS I LLHGATTASMTKPLA 1.50 (SEQID NO:1) DL03IL LYTSAALHGATTASMTKPLA 1.16 (SEQ ID NO:2) DL03KP LYTS ILLHGATTASMTAALA 1.77 (SEQ ID NO:3) DL03LA LYTS I LLHGATTASMTKPAR 1.22(SEQ ID NO:4) DL03TS LYAAI LLHGATTASMTKPLA 1.49 (SEQ ID NO:5) DL03GALYTS I LLHARTTASMTKPLA 1.67 (SEQ ID NO:6) DL03TT LYTS I LLHGAAAASMTKPLA1.43 (SEQ ID NO:7) DL03AS LYTS I LLHGATTRAMTKPLA 1.57 (SEQ ID NO:8)DL03MT LYTS I LLHGATTASAAKPLA 1.30 (SEQ ID NO:9)

[0399] The interaction of DL03wt (SEQ ID NO: 1) peptide and its mutantswith the clones identified as potential targets for the peptide DL03wt(SEQ ID NO: 1) was then quantified in a β-galactosidase YTH assay. TheYTH assay was performed in the manner described above. A general outlineof this YTH assay is illustrated in FIG. 5B. For each clone tested, aninteraction profile was developed by comparing the β-galactosidaseactivity of each mutant to that of the wild type peptide as describedabove.

[0400] Comparison of the interaction and functional profiles wasperformed by categorizing the mutants of DL03 based on the functionaland interaction assays. Based on the reporter ratio, described in panelC of FIG. 4, each mutant was categorized into one of four functionalcategories: (1) reduction of function (ROF); (2) loss of function (LOF);(3) increase of function (IOF) or (4) functionally neutral (N) ascompared to the activity of the wild type peptide, here DL03wt (SEQ IDNO: 1). As mentioned previously, cells expressing a peptide thatinhibits germline ε transcription have reporter ratios of ≧1.1. Cellexpressing a loss of function (LOF) mutant have a reporter ratio of<1.11. An increase of function mutant (IOF) shows a >50% increase inreporter ratio and a reduction of function (ROF) of mutant shows a >50%decrease in reporter ratio. Functionally neutral mutants have reporterratios that fall within ±50% of that of the DL03wt (SEQ ID NO: 1). The %increase or decrease in reporter ratio is calculated after subtracting1.0 from the individual ratios. Using these criteria, peptide DL03IL(SEQ ID NO: 2) and peptide DL03LA (SEQ ID NO: 4) were designated as ROFmutants, peptide DL03KP (SEQ ID NO: 3) was designated as a IOF mutant,peptides DL03TS (SEQ ID NO: 5), DL03GA (SEQ ID NO: 6), DL03TT (SEQ IDNO: 7), DL03AS (SEQ ID NO: 8), and DL03MT (SEQ ID NO: 9) were designatedas neutral. No LOF mutant were found for the DL03 clone.

[0401] The interaction of these IOF and ROF mutants with differentpolypeptides encoded by the putative target clones were quantified usingthe YTH assay described above. Based on the YTH assay, for each putativetarget, the mutants were categorized into the following four interactioncategories: (1) reduction of interaction (ROI); (2) loss of interaction(LOI); (3) increase of interaction (IOI); and (4) interactionallyneutral (N), by assessing the binding affinity ratio of the mutantpeptide and the wild type peptide (β-gal activity of mutant/wild type)to the putative target. For the YTH interaction assay mutants DL03IL(SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4), as well asoriginally identified peptide DL03wt (SEQ ID NO: 1) were used. Theselection criteria for categorizing the interactions is shown in FIG. 8.

[0402] Based on these functional and interaction categorizations,graphic profile representations for each potential target polypeptidewere obtained using a weighted categorization process as depicted inFIG. 9. In FIG. 9, the functional profile (reporter ratios of DL03wt(SEQ ID NO: 1) and its three mutants) is plotted along the X-axis andthe interaction profile (β-galactosidase signal from the YTH assay forDL03wt (SEQ ID NO: 1) and its three mutants) is plotted along theY-axis. The X-axis is further categorized into L (loss of function), R(reduction of function), N (functionally neutral), and I (increase offunction) based on the reporter ratios. A ratio of <1.11 is categorizedas an L and N includes ratios that fall within ±50% of the wild typepeptide ratio. A ratio that is greater than +50% of the wt peptide ratiois I. R includes ratios greater than 1.11 and ratios less than −50% ofthe wt peptide ratio. Similarly, the Y-axis is further categorized basedon the criteria in FIG. 8. In such a graphic profile, profiles thatcorrespond closely have interaction values (in this case,β-galactosidase signal) and functional values (in this case, reporterratios) that fall along a line with a positive slope. Also, closecorrespondence is indicated when the mutants fall within the samecategory type using both the reporter ratio and the β-galactosidasesignal, i.e., a mutant categorized as a L based on the reporter ratio isalso categorized as a L based on the β-galactosidase signal. In thiscase, the interaction and functional profiles for clone3.2DL3_Y_AS_U_(—)0101 fall along a line with a positive slope. Each ofthe three mutants also fall within the same respective categories basedon both reporter ratio and β-galactosidase signal. The profiles for theother putative target clones selected in the initial YTH screen do notsatisfy these criteria.

[0403] Based on the close correspondence observed between theinteraction and functional profiles, the polypeptide encoded by clone3.2DL3_Y_AS_U_(—)0101, was identified as a target for peptide DL03wt(SEQ ID NO: 1). The clone was sequenced and a sequence comparison (usinga Blast search with default parameters) with sequences in the NCBI(GenBank) nucleic database carried out. From this sequence comparisonthe clone 3.2DL3_Y_AS_U_(—)0101 was identified as human chlorideintracellular channel 1, (NCBI accession # NP_(—)001279.2;XP_(—)004183.3), also called p64CLCP.

[0404] While the invention has been described by reference to variousspecific embodiments, skilled artisans will recognize that numerousmodifications may be made thereto without departing from the spirit andthe scope of the appended claims.

[0405] All references cited throughout the disclosure are incorporatedherein by reference in their entireties for all purposes.

1 17 1 20 PRT Artificial Sequence peptide generated from a combinatoriallibrary 1 Leu Tyr Thr Ser Ile Leu Leu His Gly Ala Thr Thr Ala Ser MetThr 1 5 10 15 Lys Pro Leu Ala 20 2 20 PRT Artificial Sequence Peptidegenerated by a combinatorial library 2 Leu Tyr Thr Ser Ala Ala Leu HisGly Ala Thr Thr Ala Ser Met Thr 1 5 10 15 Lys Pro Leu Ala 20 3 20 PRTArtificial Sequence peptide generated from combinatorial library 3 LeuTyr Thr Ser Ile Leu Leu His Gly Ala Thr Thr Ala Ser Met Thr 1 5 10 15Ala Ala Leu Ala 20 4 20 PRT Artificial Sequence peptide generated fromcombinatorial library 4 Leu Tyr Thr Ser Ile Leu Leu His Gly Ala Thr ThrAla Ser Met Thr 1 5 10 15 Lys Pro Ala Arg 20 5 20 PRT ArtificialSequence peptide generated from combinatorial library 5 Leu Tyr Ala AlaIle Leu Leu His Gly Ala Thr Thr Ala Ser Met Thr 1 5 10 15 Lys Pro LeuAla 20 6 20 PRT Artificial Sequence peptide generated from combinatoriallibrary 6 Leu Tyr Thr Ser Ile Leu Leu His Ala Arg Thr Thr Ala Ser MetThr 1 5 10 15 Lys Pro Leu Ala 20 7 20 PRT Artificial Sequence peptidegenerated from combinatorial library 7 Leu Tyr Thr Ser Ile Leu Leu HisGly Ala Ala Ala Ala Ser Met Thr 1 5 10 15 Lys Pro Leu Ala 20 8 20 PRTArtificial Sequence Peptide generated from combinatorial library 8 LeuTyr Thr Ser Ile Leu Leu His Gly Ala Thr Thr Arg Ala Met Thr 1 5 10 15Lys Pro Leu Ala 20 9 20 PRT Artificial Sequence Peptide generated fromcombinatorial library 9 Leu Tyr Thr Ser Ile Leu Leu His Gly Ala Thr ThrAla Ser Ala Ala 1 5 10 15 Lys Pro Leu Ala 20 10 241 PRT Homo sapiens 10Met Ala Glu Glu Gln Pro Gln Val Glu Leu Phe Val Lys Ala Gly Ser 1 5 1015 Asp Gly Ala Lys Ile Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Met 20 2530 Val Leu Trp Leu Lys Gly Val Thr Phe Asn Val Thr Thr Val Asp Thr 35 4045 Lys Arg Arg Thr Glu Thr Val Gln Lys Leu Cys Pro Gly Gly Gln Leu 50 5560 Pro Phe Leu Leu Tyr Gly Thr Glu Val His Thr Asp Thr Asn Lys Ile 65 7075 80 Glu Glu Phe Leu Glu Ala Val Leu Cys Pro Pro Arg Tyr Pro Lys Leu 8590 95 Ala Ala Leu Asn Pro Glu Ser Asn Thr Ala Gly Leu Asp Ile Phe Ala100 105 110 Lys Phe Ser Ala Tyr Ile Lys Asn Ser Asn Pro Ala Leu Asn AspAsn 115 120 125 Leu Glu Lys Gly Leu Leu Lys Ala Leu Lys Val Leu Asp AsnTyr Leu 130 135 140 Thr Ser Pro Leu Pro Glu Glu Val Asp Glu Thr Ser AlaGlu Asp Glu 145 150 155 160 Gly Val Ser Gln Arg Lys Phe Leu Asp Gly AsnGlu Leu Thr Leu Ala 165 170 175 Asp Cys Asn Leu Leu Pro Lys Leu His IleVal Gln Val Val Cys Lys 180 185 190 Lys Tyr Arg Gly Phe Thr Ile Pro GluAla Phe Arg Gly Val His Arg 195 200 205 Tyr Leu Ser Asn Ala Tyr Ala ArgGlu Glu Phe Ala Ser Thr Cys Pro 210 215 220 Asp Asp Glu Glu Ile Glu LeuAla Tyr Glu Gln Val Ala Lys Ala Leu 225 230 235 240 Lys 11 603 DNA Homosapiens 11 ctcgaggaca gtgacctggg agtgagtaca aggtgaggcc accactcagggtgccagctc 60 caagcgggtc acagggacga gggctgcggc catcaggagg ccctgcacacacatctggga 120 cacgcgcccc cgagggccag ttcacctcag tgcgcctcat tctcctgcacaaaagcgccc 180 ccatcctttc ttcacaaggc tttcgtggaa gcagaggcgt cgatgcccagtaccctctcc 240 ctttcccagg caacgggacc ccaagtttgc tgactgggac caccaagccacgcatgcgtc 300 aagagtgaga gtccgggacc taggcagggg ccctggggtt gggcctgagagagaagagaa 360 cctcccccag cactcggtgt gcatcggtag tgaaggagcc tcacctgacccccgctgttg 420 ctcaatcgac ttcccaagaa cagagagaaa agggaacttc cagggcggcccgggcctcct 480 gggggttccc accccatttt tagctgaaag cactgaggca gagctccccctacccaggct 540 ccactgcccg gcacagaaat aacaaccacg gttactgatc atctgggagctgtccaggaa 600 ttc 603 12 20 DNA Artificial Sequence pcr primer 12atccacaggc accaaatgga 20 13 19 DNA Artificial Sequence pcr primer 13ggaagacgga tgggctctg 19 14 20 DNA Artificial Sequence DNA probe 14acccggcgct tcagcctcca 20 15 15 DNA Artificial Sequence pcr primer 15cagcactgcg ggccc 15 16 18 DNA Artificial Sequence pcr primer 16tcagcgggaa gaccttgg 18 17 22 DNA Artificial Sequence DNA probe 17ccagcagcct gaccagcatc cc 22

What is claimed is:
 1. A method of identifying a compound that modulatesIL-4 receptor-mediated IgE production or a process associated therewith,comprising determining whether the compound binds a CLIC1, wherein theability to bind the CLIC1 identifies the compound as being a modulatorof IL-4 induced IgE production or a process associated therewith.
 2. Themethod of claim 1 in which the compound identified inhibits IL-4receptor-mediated IgE production.
 3. The method of claim 1 in which thecompound identified inhibits IL-4 receptor-mediated isotype switching ofB-cells.
 4. The method of claim 1 in which the compound identifiedinhibits IL-4 induced transcription of a germline ε promoter.
 5. Themethod of claim 1 in which the compound specifically modulates IL-4induced IgE production or a process associated therewith.
 6. The methodof claim 1 in which the CLIC1 is a mammalian CLIC1.
 7. The method ofclaim 6 in which the mammalian CLIC1 is a human CLIC1.
 8. The method ofclaim 1 in which it is determined whether the compound binds the CLIC1in a competitive binding assay.
 9. The method of claim 8 in which thecompound competes for binding the CLIC1 with an active DL03 compound.10. The method of claim 9 in which the active DL03 compound is selectedfrom the group consisting of DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO:2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4), DL03TS (SEQ ID NO: 5),DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7), DL03AS (SEQ ID NO: 8),DL03MT (SEQ ID NO: 9) and an analog thereof.
 11. The method of claim 1which is carried out in a cell-free system with an isolated CLIC1. 12.The method of claim 1 in which the compound is a small organic compound.13. The method of claim 12 in which the small organic compound has amolecular weight in the range of about 100-2500 Dalton.
 14. The methodof claim 1 in which the compound is selected from the group consistingof peptides and peptide analogs.
 15. The method of claim 1, furtherincluding the step of confirming that the identified compound modulatesIL-4 induced IgE production or a process associated therewith.
 16. Amethod of identifying compounds that modulate IL-4 receptor-mediated IgEproduction or a process associated therewith, comprising the step ofcontacting a compound from pool of candidate compounds with a CLIC1 andidentifying those compounds of the pool that bind the CLIC1, therebyidentifying those compounds of the pool that modulate IL-4receptor-mediated IgE production or a process associated therewith. 17.The method of claim 16 which is carried out in the presence of acompound known to bind the CLIC1 such that those compounds of the poolthat competitively bind the CLIC1 are identified.
 18. The method ofclaim 17 in which the compound known to bind the CLIC1 is an active DL03compound.
 19. The method of claim 18 in which the active DL03 compoundis selected from the group consisting of DL03wt (SEQ ID NO: 1), DL03IL(SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4), DL03TS(SEQ ID NO: 5), DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7), DL03AS(SEQ ID NO: 8), DL03MT (SEQ ID NO: 9) and an analog thereof.
 20. Themethod of claim 16 in which the candidate compounds are peptides orsmall organic compounds.
 21. The method of claim 20 in which the pool ofcandidate compounds is a phage display library.
 22. The method of claim20 in which the candidate compounds are immobilized on a substrate or aplurality of substrates.
 23. The method of claim 16 in which thecompound or CLIC1 is labeled with a detectable label.
 24. The method ofclaim 16 in which the CLIC1 is immobilized on a substrate.
 25. Acompound comprising a peptide or peptide analog, or a pharmaceuticallyacceptable salt thereof, having the formula (I):Z¹-X¹˜X²˜X³˜X⁴˜X⁵˜X⁶˜X⁷˜X⁸˜X⁹˜X¹⁰˜X¹¹˜X¹²˜X¹³˜X¹⁴˜X¹⁵˜X¹⁶˜X¹⁷˜X¹⁸˜X¹⁹˜X²⁰-Z²wherein: X¹ is an aliphatic residue; X² is an aromatic residue; X³ is asmall polar or small aliphatic residue; X⁴ is a small polar or smallaliphatic residue; X⁵ is an aliphatic or nonpolar residue; X⁶ is analiphatic or nonpolar residue; X⁷ is an aliphatic or nonpolar residue;X⁸ is an aromatic residue; X⁹ is small nonpolar residue; X¹⁰ is analiphatic or basic residue; X¹¹ is a small polar or small aliphaticresidue; X¹² is a small polar or small aliphatic residue; X¹³ is a smallaliphatic or basic residue; X¹⁴ is a small polar or small aliphaticresidue; X¹⁵ is a nonpolar or small aliphatic residue; X¹⁶ is a smallpolar or small aliphatic residue; X¹⁷ is a small aliphatic or basicresidue; X¹⁸ is a small aliphatic or conformationally-constrainedresidue; X¹⁹ is a small aliphatic or aliphatic residue; and X²⁰ is asmall aliphatic or basic residue; Z¹ is RRN—, RC(O)NR—, RS(O)₂NR— or anamino-terminal blocking group; Z² is —C(O)OR, —C(O)O—, —C(O)NRR or acarboxyl-terminal blocking group; each R is independently selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl and substituted heteroarylalkyl;each “˜” independently represents an amide, a substituted amide or anisostere of an amide; each “—” represents a bond, or a 1 to 10 residuepeptide or peptide analog; and wherein one or more of X¹, X², X¹⁹, orX²⁰ may be absent.
 26. The compound of claim 25 in which each “˜” is anamide, Z¹ is H₂N— and Z² is —C(O)OH or C(O)O⁻.
 27. The compound of claim25 having the formula: Leu Tyr X³X⁴X⁵X⁶Leu HisX⁹X¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹X²⁰ wherein: X³ is a small polar orsmall aliphatic residue; X⁴ is a small polar or small aliphatic residue;X⁵ is an aliphatic residue; X⁶ is an aliphatic residue; X⁹ is smallnonpolar residue; X¹⁰ is an aliphatic or basic residue; X¹¹ is a smallpolar or small aliphatic residue; X¹² is a small polar or smallaliphatic residue; X¹³ is a small aliphatic or basic residue; X¹⁴ is asmall polar or small aliphatic residue; X¹⁵ is a nonpolar or smallaliphatic residue; X¹⁶ is a small polar or small aliphatic residue; X¹⁷is a small aliphatic or a basic residue; X¹⁸ is a small aliphatic or aconformationally-constrained residue; X¹⁹ is a small aliphatic or analiphatic residue; and X²⁰ is a small aliphatic or a basic residue. 28.The compound of claim 27 in which X⁵ is Ile or Ala and/or X⁶ is Leu orAla.
 29. The compound of claim 27 in which X⁹ is Gly or Ala and/or X¹⁰is Arg or Ala.
 30. The compound of claim 27 in which X¹¹ is Thr or Alaand/or X¹² is Thr or Ala.
 31. The compound of claim 27 in which X¹³ isArg or Ala and/or X¹⁴ is Ser or Ala.
 32. The compound of claim 27 inwhich X¹⁵ is Met or Ala and/or X¹⁶ is Thr or Ala.
 33. The compound ofclaim 27 in which X¹⁷ is Lys or L-Ala and/or X¹⁸ is Pro or Ala.
 34. Thecompound of claim 27 in which X¹⁹ is Leu or Ala and/or X²⁰ is Arg orAla.
 35. The compound of claim 25 which is selected from the groupconsisting of DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO: 2), DL03KP (SEQID NO: 3), DL03LA (SEQ ID NO: 4) DL03TS (SEQ ID NO: 5), DL03GA (SEQ IDNO: 6), DL03TT (SEQ ID NO: 7), DL03AS (SEQ ID NO: 8), DL03MT (SEQ ID NO:9) and an analog thereof.
 36. A compound comprising a peptide or peptideanalog having the formula (IV)Z¹-L˜Y˜T˜S˜L˜L˜H˜G˜A˜T˜T˜A˜S˜M˜T˜K˜P˜L˜A-Z²   (IV) wherein: Z¹ is RRN—,RC(O)NR—, RS(O)₂NR— or an amino-terminal blocking group; Z² is —C(O)OR,—C(O)O—, —C(O)NRR or a carboxyl-terminal blocking group; each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl andsubstituted heteroarylalkyl; each “˜” independently represents an amide,a substituted amide or an isostere of an amide; and each “—” representsa bond, or a 1 to 10 residue peptide or peptide analog; and variantsthereof in which 1, 2, 3, or 4 of the amino acid residues set forth inIV are replaced by another amino acid selected from the same class asthe original amino acid or by an Ala or a Arg residue.
 37. A compoundidentified by the method of claim 1
 38. A pharmaceutical compositioncomprising a compound according to claim 25 or claim 36 and apharmaceutically acceptable carrier, excipient or diluent.
 39. Apharmaceutical composition comprising a compound identified by themethod of claim 1 and a pharmaceutically acceptable carrier, excipientor diluent.
 40. A method of modulating IL-4 receptor-mediated IgEproduction in a cell, comprising administering to the cell an effectiveamount of a compound that binds a CLIC1.
 41. The method of claim 40 inwhich the compound competitively binds the CLIC1 in the presence of anactive DL03 compound.
 42. The method of claim 40 in which the compoundis selected from the group consisting of DL03wt (SEQ ID NO: 1), DL03IL(SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4) DL03TS (SEQID NO: 5), DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7), DL03AS (SEQ IDNO: 8), DL03MT (SEQ ID NO: 9) and an analog thereof.
 43. The method ofclaim 40 in which the compound is identified by the method of claim 1.44. A method of treating an animal suffering from a diseasecharacterized by, caused by or associated with IgE production and/oraccumulation and/or symptoms associated therewith, comprisingadministering to the animal an amount of a compound that binds a CLIC1effective to treat the disease.
 45. The method of claim 44 in which thecompound competitively binds the CLIC1 in the presence of an active DL03compound.
 46. The method of claim 44 in which the compound is selectedfrom the group consisting of DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO:2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4) DL03TS (SEQ ID NO: 5),DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7), DL03AS (SEQ ID NO: 8),DL03MT (SEQ ID NO: 9) and an analog thereof.
 47. The method of claim 44in which the compound is identified by the method of claim
 1. 48. Themethod of claim 44 in which the disease is selected from the groupconsisting of an allergy, an atopic disorder, allergic rhinitis,allergic conjunctivitis, systemic mastocytosis, hyper IgE syndrome, IgEgammopathies and B-cell lymphoma.
 49. The method of claim 48 in whichthe allergy is an anaphylactic allergic reaction.
 50. The method ofclaim 48 in which the atopic disorder is selected from the groupconsisting of atopic dermatitis, atopic eczema and atopic asthma.
 51. Akit for identifying compounds that modulate IL-4 induced IgE production,comprising a CLIC1 or a cell expressing a CLIC1 and a compound thatcompetitively binds the CLIC1 in the presence of an active DL03compound.
 52. The kit of claim 51 in which the compound is selected fromthe group consisting of DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO: 2),DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4) DL03TS (SEQ ID NO: 5),DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7), DL03AS (SEQ ID NO: 8),DL03MT (SEQ ID NO: 9) and an analog thereof.
 53. A method of inhibitinggermline ε transcription in a cell, comprising administering to the cellan effective amount of a compound that binds a CLIC1.
 54. The method ofclaim 53 in which the compound competitively binds the CLIC1 in thepresence of an active DL03 compound.
 55. The method of claim 53 in whichthe compound is selected from the group consisting of DL03wt (SEQ ID NO:1), DL03IL (SEQ ID NO: 2), DL03KP (SEQ ID NO: 3), DL03LA (SEQ ID NO: 4)DL03TS (SEQ ID NO: 5), DL03GA (SEQ ID NO: 6), DL03TT (SEQ ID NO: 7),DL03AS (SEQ ID NO: 8), DL03MT (SEQ ID NO: 9) and an analog thereof. 56.The method of claim 54 in which the active DL03 compound is selectedfrom DL03wt (SEQ ID NO: 1), DL03IL (SEQ ID NO: 2), DL03KP (SEQ ID NO:3), DL03LA (SEQ ID NO: 4) DL03TS (SEQ ID NO: 5), DL03GA (SEQ ID NO: 6),DL03TT (SEQ ID NO: 7), DL03AS (SEQ ID NO: 8), DL03MT (SEQ ID NO: 9) andan analog thereof.
 57. The method of any one of claim 1, claim 16, claim40, claim 44 and claim 53, in which the CLIC1 is a polypeptide havingthe amino acid sequence of SEQ ID NO: 10.